U.S. patent number 10,729,771 [Application Number 16/076,971] was granted by the patent office on 2020-08-04 for anti-lam and anti-pim6/lam monoclonal antibodies for diagnosis and treatment of mycobacterium tuberculosis infections.
This patent grant is currently assigned to RUTGERS, THE STATE UNIVERSITY OF NEW JERSEY. The grantee listed for this patent is Rutgers, The State University of New Jersey. Invention is credited to Alok Choudhary, Abraham Pinter.
View All Diagrams
United States Patent |
10,729,771 |
Pinter , et al. |
August 4, 2020 |
Anti-LAM and anti-PIM6/LAM monoclonal antibodies for diagnosis and
treatment of Mycobacterium tuberculosis infections
Abstract
The present invention broadly provides different compositions,
kits, vectors, and methods including monoclonal antibodies directed
to epitopes found within lipoarabinomannan (LAM) and
phosphatidyl-myo-inositol mannoside 6 (PIM6) for the diagnosis and
treatment of Mycobacterium tuberculosis infections.
Inventors: |
Pinter; Abraham (Brooklyn,
NY), Choudhary; Alok (Newark, NJ) |
Applicant: |
Name |
City |
State |
Country |
Type |
Rutgers, The State University of New Jersey |
New Brunswick |
NJ |
US |
|
|
Assignee: |
RUTGERS, THE STATE UNIVERSITY OF
NEW JERSEY (New Brunswick, NJ)
|
Family
ID: |
1000004962165 |
Appl.
No.: |
16/076,971 |
Filed: |
February 1, 2017 |
PCT
Filed: |
February 01, 2017 |
PCT No.: |
PCT/US2017/016058 |
371(c)(1),(2),(4) Date: |
August 09, 2018 |
PCT
Pub. No.: |
WO2017/139153 |
PCT
Pub. Date: |
August 17, 2017 |
Prior Publication Data
|
|
|
|
Document
Identifier |
Publication Date |
|
US 20190038747 A1 |
Feb 7, 2019 |
|
Related U.S. Patent Documents
|
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
|
62293406 |
Feb 10, 2016 |
|
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K
39/04 (20130101); A61K 39/40 (20130101); C07K
16/1289 (20130101); G01N 33/5695 (20130101); A61P
31/06 (20180101); G01N 33/56933 (20130101); G01N
2333/35 (20130101); C07K 2317/21 (20130101); G01N
2800/44 (20130101); C07K 2317/622 (20130101); C07K
2317/92 (20130101); G01N 2400/02 (20130101) |
Current International
Class: |
G01N
33/569 (20060101); A61K 39/40 (20060101); C07K
16/12 (20060101); A61P 31/06 (20060101); A61K
39/04 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
Kussie et al (Journal of Immunology, 152:146-152, 1994). cited by
examiner .
Chen et al, (The EMBO Journal, 14(12):2784-2794, 1995). cited by
examiner .
Rudikoff et al PNAS 79:1979-1983, 1982. cited by examiner .
Bendig (Methods: A Companion to Methods in Enzymology 1995;
8:83-93). cited by examiner .
Paul, Fundamental Immunology, 3rd Edition, 1993, pp. 292-295, under
the heading Fv Structure and Diversity in Three Dimensions. cited
by examiner .
MacCallum et al. J. Mol. Biol. (1996) 262,732-745. cited by
examiner .
Pascalis et al. The Journal of Immunology (2002) 169, 3076-3084.
cited by examiner .
Casset et al. (2003) BBRC 307, 198-205. cited by examiner .
Vajdos et al. (2002) 320, 415-428. cited by examiner .
Chen et al. J. Mol. Bio. (1999) 293, 865-881. cited by examiner
.
Wu et al. J. Mol. Biol. (1999) 294, 151-162. cited by examiner
.
Mikayama et al. (Nov.1993. Proc.Natl.Acad.Sci. USA, vol. 90 :
10056-10060). cited by examiner .
Torrelles et al (J Biol Chem. Sep. 24, 2004;279(39):41227-39. cited
by examiner .
Rademacher et al., "Ligand Specificity of CS-35, a Monoclonal
Antibody That Recognizes Mycobacterial Lipoarabinomannan: A Model
System for Oligofuranoside--Protein Recognition," J. Am. Chem. Soc.
(2007); 129:10489-10502. cited by applicant .
Chan et al., "The diagnositc targeting of a carbohydrate virulence
factor from M. Tuberculosis," Scientific Reports (May 15, 2015);
5(1):1-12. cited by applicant .
Choudhary et al., "Characterization of the Antigenic Heterogeneity
of Lipoarabinomannan, the Major Surface Glycolipid of Mycobacterium
tuberculosis, and Complexity of Antibody Specificities toward This
Antigen," The Journal of Immunology (2018); 200:3053-3066. cited by
applicant.
|
Primary Examiner: Graser; Jennifer E
Attorney, Agent or Firm: Fox Rothschild LLP
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATION
This application claims priority to U.S. Provisional Application
No. 62/293,406 filed Feb. 10, 2016, which is incorporated herein by
reference in its entirety.
Claims
What is claimed:
1. A human monoclonal anti-lipoarabinomannan (anti-LAM) antibody,
or an antigen-binding portion thereof, that specifically binds to a
LAM epitope comprising an Ara4 structure, an Ara6 structure, or a
combination thereof, wherein the anti-LAM antibody comprises a CDR1
light chain variable region having at least 80% identity with SEQ
ID NO: 1 or antigenic fragments thereof, a CDR2 light chain
variable region having at least 80% identity with SEQ ID NO: 2 or
antigenic fragments thereof, a CDR3 light chain variable region
having at least 80% identity with SEQ ID NO: 3 or SEQ ID NO: 26 or
antigenic fragments thereof, a CDR1 heavy chain variable region
having at least 80% identity with SEQ ID NO: 4 or antigenic
fragments thereof, a CDR2 heavy chain variable region having at
least 80% identity with SEQ ID NO: 5 or antigenic fragments
thereof, and a CDR3 heavy chain variable region having at least 80%
identity with SEQ ID NO: 6 or SEQ ID NO: 23 or antigenic fragments
thereof.
2. The human monoclonal anti-LAM antibody or antigen-binding
portion thereof of claim 1, said antibody comprising a heavy chain
variable region comprising the amino acid sequences of SEQ ID NO:21
and SEQ ID NO:23, and a light chain variable region comprising the
amino acid sequences of SEQ ID NO: 24 and SEQ ID NO:26.
3. The human monoclonal anti-LAM antibody or antigen-binding
portion thereof of claim 1, wherein the anti-LAM antibody is an
scFv-IgG, an IgA or an IgM antibody.
Description
FIELD OF THE INVENTION
Compositions, kits, vectors, and methods including antibodies
directed to epitopes found within lipoarabinomannan (LAM)
lipomannan (LM) and phosphatidyl-myo-inositol mannoside 6 (PIM6)
for the diagnosis, prevention and treatment of Mycobacterium
tuberculosis infections are described herein.
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been
submitted in ASCII format via EFS-WEB and is hereby incorporated by
reference in its entirety. Said ASCII copy, created on Feb. 1,
2017, is named 096747.00337_ST25.txt and is 29,097 bytes in
size.
BACKGROUND
A. Mycobacterium tuberculosis
Tuberculosis (TB) remains one of the world's deadliest communicable
diseases, currently infecting approximately 1/3 of the world's
population. According to the WHO Global Tuberculosis Report, 2014:
Tuberculosis, in 2013, an estimated 9.0 million people developed
TB, and 1.5 million died from the disease. Although there currently
are effective drugs available for TB, these require lengthy
treatments with multiple antibiotics, and are increasingly
compromised by the development of multi-drug resistant (MDR-TB)
strains, which currently are responsible for about 3.5% of recent
infections. These strains are much harder to treat and have
significantly poorer cure rates. Also spreading are extensively
drug-resistant TB (XDR-TB) strains, which are even more expensive
and difficult to treat than MDR-TB strains, and have now been
reported in 100 countries around the world. Consequently, new
approaches are needed for the earlier diagnosis and treatment of TB
infections.
B. Lipoarabinomannan (LAM)
The glycolipid lipoarabinomannan (LAM) is a major structural and
antigenic component of the cell wall of members of the
Mycobacterium tuberculosis-complex, and it mediates a number of
important functions that promote productive infection and disease
development. LAM is also an important immunodiagnostic target for
detecting active infection with TB, especially in patients
co-infected with HIV-1, and a potential vaccine target. Despite the
importance of LAM as an immunodiagnostic target and its significant
role in the physiology of M.tb infection and pathogenicity,
surprisingly little is known about the nature of the human humoral
response towards this antigen. Previously available LAM-specific
monoclonal antibodies have been derived from mice immunized with
LAM purified from either Mycobacterium leprae or Mycobacterium
tuberculosis, and there have been no descriptions of any human
monoclonal antibodies against LAM that have been induced in
response either to immunization or to infection by Mycobacterium
tuberculosis.
Lipomannan (LM)--is the immediate precursor to LAM and contain a
phosphatidyl-myo-inositol domain modified by a mannan domain
comprised of an .alpha.(1.fwdarw.6)-linked Manp backbone
substituted with short .alpha.(1.fwdarw.2)-mannopyranosyl side
chains, but with no arabinose side chains.
C. Phosphatidyl-Myo-Inositol Mannoside 6 (PIM6)
PIM6 is a product of PIM2, a common precursor to LM and LAM. The
core of these molecules is a myo-inositol structure glycosylated
with a Manp unit at positions 2 and 6. In PIM6, the Manp unit at
positions 6 is further substituted by two terminal
.alpha.-Manp(1.fwdarw.2)-linked sugars identical to the mannose cap
on ManLAM. These molecules are acylated by as many as 4 fatty acid
chains, attached to the inositol head group and to the core Man
residue, which non-covalently anchor these molecules to the inner
and outer membranes of the cell envelope. PIM6 was reported to bind
to C-type lectins and DC-SIGN, the major receptor on dendritic
cells, and to be a strong TLR2 agonist and enhancer of HIV-1
replication that possesses potent anti-inflammatory activities.
SUMMARY OF THE INVENTION
Described herein are novel anti-LAM and anti-PIM6/LAM monoclonal
antibodies (mAbs) for diagnosis and treatment of Mycobacterium
tuberculosis infections. The isolation and characterization of
these novel human antibodies specific for glycolipids of
Mycobacterium tuberculosis, including human mAbs specific for LAM
epitopes, and a human mAb specific for an epitope shared by LAM and
PIM6, are described below.
Accordingly, described herein is a human monoclonal
anti-lipoarabinomannan (anti-LAM) antibody, or an antigen-binding
portion thereof, that specifically binds to a LAM epitope including
an Ara4 structure, an Ara6 structure, or a combination thereof,
wherein the anti-LAM antibody includes a CDR1 variable light region
having at least 80% identity with SEQ ID NO: 1 or antigenic
fragments thereof, a CDR2 variable light region having at least 80%
identity with SEQ ID NO: 2 or antigenic fragments thereof, a CDR3
variable light region having at least 80% identity with SEQ ID NO:
3 or SEQ ID NO: 26 or antigenic fragments thereof, a CDR1 variable
heavy region having at least 80% identity with SEQ ID NO: 4 or
antigenic fragments thereof, a CDR2 variable heavy region having at
least 80% identity with SEQ ID NO: 5 or antigenic fragments
thereof, and a CDR3 variable heavy region having at least 80%
identity with SEQ ID NO: 6 or SEQ ID NO: 23 or antigenic fragments
thereof. The human monoclonal anti-LAM antibody or antigen-binding
portion thereof can include a heavy chain variable region including
the amino acid sequences of SEQ ID NO:21 and SEQ ID NO:23, and a
light chain variable region including the amino acid sequences of
SEQ ID NO: 24 and SEQ ID NO:26. The anti-LAM antibody can be an
scFv-IgG, and IgGa or an IgM antibody. An example of an ant-LAM
antibody is A194.
Also described herein is a human monoclonal anti-LAM antibody or an
antigen-binding portion thereof, that specifically binds to a LAM
epitope including at least one of: a mannose-capped Ara4 structure
and a mannose-capped Ara6 structure. The anti-LAM antibody can
include a CDR1 variable light region having at least 80% identity
with SEQ ID NO: 7 or antigenic fragments thereof, a CDR2 variable
light region having at least 80% identity with SEQ ID NO: 8 or
antigenic fragments thereof, a CDR3 variable light region having at
least 80% identity with SEQ ID NO: 9 or SEQ ID NO: 32 or antigenic
fragments thereof, a CDR1 variable heavy region having at least 80%
identity with SEQ ID NO: 10 or antigenic fragments thereof, a CDR2
variable heavy region having at least 80% identity with SEQ ID NO:
11 or antigenic fragments thereof, and a CDR3 variable heavy region
having at least 80% identity with SEQ ID NO: 12 or SEQ ID NO: 29 or
antigenic fragments thereof. The antibody can include a heavy chain
variable region including the amino acid sequence of SEQ ID NO:43
and a light chain variable region including the amino acid sequence
of SEQ ID NO:44. The anti-LAM antibody can be, for example, an IgM
or IgA antibody. An example of an anti-LAM antibody is P3B09.
Further described herein is a human monoclonal anti-LAM antibody,
or an antigen-binding portion thereof, that specifically binds to a
LAM epitope including an .alpha.-Manp(1.fwdarw.2) linked structure
attached at a nonreducing end of Ara4 or Arab, wherein the anti-LAM
antibody includes a CDR1 variable light region having at least 80%
identity with SEQ ID NO: 7 or antigenic fragments thereof, a CDR2
variable light region having at least 80% identity with SEQ ID NO:
8 or antigenic fragments thereof, a CDR3 variable light region
having at least 80% identity with SEQ ID NO: 9 or antigenic
fragments thereof, a CDR1 variable heavy region having at least 80%
identity with SEQ ID NO: 10 or antigenic fragments thereof, a CDR2
variable heavy region having at least 80% identity with SEQ ID NO:
11 or antigenic fragments thereof, and a CDR3 variable heavy region
having at least 80% identity with SEQ ID NO: 12 or antigenic
fragments thereof. The anti-LAM antibody (e.g., P3B09) can be, for
example, an IgM or IgA antibody.
Yet further described herein is a human monoclonal anti-PIM6/LAM
antibody, or an antigen-binding portion thereof, that specifically
binds to an epitope present in LAM and PIM6, the epitope including
at least one polymannose structure. The epitope is in the PIM6
mannan domain, and is also present in mycobacterial lipomannan
(LM). The anti-PIM6/LAM antibody can include a CDR1 variable light
region having at least 80% identity with SEQ ID NO: 13 or antigenic
fragments thereof, a CDR2 variable light region having at least 80%
identity with SEQ ID NO: 14 or antigenic fragments thereof, a CDR3
variable light region having at least 80% identity with SEQ ID NO:
15 or antigenic fragments thereof, a CDR1 variable heavy region
having at least 80% identity with SEQ ID NO: 16 or antigenic
fragments thereof, a CDR2 variable heavy region having at least 80%
identity with SEQ ID NO: 17 or antigenic fragments thereof, and a
CDR3 variable heavy region having at least 80% identity with SEQ ID
NO: 18 or antigenic fragments thereof. The antibody can, for
example, include a heavy chain variable region including the amino
acid sequence of SEQ ID NO:47 and a light chain variable region
including the amino acid sequence of SEQ ID NO:48. The
anti-PIM6/LAM antibody can be, for example, an IgM, IgA or IgG
antibody. An example of an anti-PIM6/LAM antibody is P95C1.
Also described herein is a kit for detecting at least one LAM
epitope. The kit includes (a) at least a first anti-LAM antibody
that binds specifically to a LAM epitope; (b) a support to which
the at least first anti-LAM antibody is bound; (c) a detection
antibody that binds specifically to LAM, or specifically to the at
least first anti-LAM antibody, wherein the detection antibody is
labeled with a reporter molecule; and (d) a buffer. The at least
first anti-LAM antibody is, for example, a human monoclonal
anti-LAM antibody as described herein. The detection antibody can
be, for example, a second anti-LAM antibody that binds specifically
to LAM. In some embodiments, the at least one of the first anti-LAM
antibody and the second anti-LAM antibody is an scFv-IgG or IgM
antibody and includes a CDR1 variable light region having at least
80% identity with SEQ ID NO: 1 or antigenic fragments thereof, a
CDR2 variable light region having at least 80% identity with SEQ ID
NO: 2 or antigenic fragments thereof, a CDR3 variable light region
having at least 80% identity with SEQ ID NO: 3 or SEQ ID NO: 26 or
antigenic fragments thereof, a CDR1 variable heavy region having at
least 80% identity with SEQ ID NO: 4 or antigenic fragments
thereof, a CDR2 variable heavy region having at least 80% identity
with SEQ ID NO: 5 or antigenic fragments thereof, and a CDR3
variable heavy region having at least 80% identity with SEQ ID NO:
6 or SEQ ID NO:23 or antigenic fragments thereof. In some
embodiments of the kit, at least one of the first anti-LAM antibody
and the second anti-LAM antibody includes a heavy chain variable
region including the amino acid sequences of SEQ ID NO:21 and SEQ
ID NO:23, and a light chain variable region including the amino
acid sequences of SEQ ID NO: 24 and SEQ ID NO:26.
Still further described herein is a method of diagnosing an active
tuberculosis infection in an individual including: (a) obtaining a
sample from an individual that includes or is suspected of
including LAM; (b) treating said sample to expose individual LAM
epitopes; (c) contacting said sample with at least a first antibody
that binds specifically to a first epitope on said LAM; (d)
contacting said sample with a detection antibody that binds
specifically to LAM, or specifically to the at least first
antibody; (e) detecting binding of the at least first antibody to
said first epitope on LAM; and (f) diagnosing said patient as
having an active tuberculosis infection, the binding of the at
least first antibody to said first epitope on LAM indicating an
active tuberculosis infection. The at least first antibody is, for
example, a human monoclonal anti-LAM antibody or human monoclonal
anti-PIM6/LAM antibody as described herein. The detection antibody
can be, for example, an anti-LAM antibody that binds specifically
to LAM. In some embodiments of the method, the at least first
antibody and the detection antibody each include a CDR1 variable
light region having at least 80% identity with SEQ ID NO: 1 or
antigenic fragments thereof, a CDR2 variable light region having at
least 80% identity with SEQ ID NO: 2 or antigenic fragments
thereof, a CDR3 variable light region having at least 80% identity
with SEQ ID NO: 3 or SEQ ID NO: 26 or antigenic fragments thereof,
a CDR1 variable heavy region having at least 80% identity with SEQ
ID NO: 4 or antigenic fragments thereof, a CDR2 variable heavy
region having at least 80% identity with SEQ ID NO: 5 or antigenic
fragments thereof, and a CDR3 variable heavy region having at least
80% identity with SEQ ID NO: 6 or SEQ ID NO: 23 or antigenic
fragments thereof. In some embodiments of the method, at least one
of the first antibody and the detection antibody is an scFv-IgG or
IgM antibody and includes a CDR1 region having a variable light
region having at least 80% identity with SEQ ID NO: 1 or antigenic
fragments thereof, a CDR2 variable light region having at least 80%
identity with SEQ ID NO: 2 or antigenic fragments thereof, a CDR3
variable light region having at least 80% identity with SEQ ID NO:
3 or SEQ ID NO: 26 or antigenic fragments thereof, a CDR1 variable
heavy region having at least 80% identity with SEQ ID NO: 4 or
antigenic fragments thereof, a CDR2 variable heavy region having at
least 80% identity with SEQ ID NO: 5 or antigenic fragments
thereof, and a CDR3 variable heavy region having at least 80%
identity with SEQ ID NO: 6 or SEQ ID NO: 23 or antigenic fragments
thereof. In some embodiments, the individual is a human.
Also described herein is a method of treating a tuberculosis
infection in an individual (e.g., a human). The method includes
administering to said individual a therapeutically effective amount
of at least one human monoclonal anti-LAM antibody or human
monoclonal anti-PIM6/LAM antibody as described herein. The method
can further include administering to said individual a
therapeutically effective amount of at least one antibiotic. The
tuberculosis infection can be a multi-drug resistant (MDR-TB)
tuberculosis infection.
Further described herein are nucleotide sequences encoding the
heavy chains and light chains (including variable regions) of the
antibodies described herein.
A. Anti-LAM Antibodies and Anti-PIM6/LAM Antibodies
In some embodiments, the invention provides an anti-LAM antibody,
or an antigen binding portion thereof. In some embodiments, the
invention provides an anti-PIM6/LAM antibody, or an antigen binding
portion thereof. An anti-LAM antibody (or antigen binding portion
thereof) as described herein binds specifically to a LAM epitope.
An anti-PIM6/LAM antibody (or antigen binding portion thereof) as
described herein binds specifically to both a LAM epitope and a
PIM6 epitope. In some embodiments, the LAM and PIM6 epitopes are
derived from various mycobacterial species. In further embodiments,
the various mycobacterial species are virulent members of the
Mycobacterium tuberculosis-complex. In yet further embodiments, the
mycobacterial species is Mycobacterium tuberculosis. In some
embodiments, the anti-LAM antibody or anti-PIM6/LAM antibody is a
monoclonal antibody (mAb). In further embodiments, the anti-LAM
antibody or anti-PIM6/LAM antibody is a human monoclonal anti-LAM
antibody or human monoclonal anti-PIM6/LAM antibody, respectively.
In other embodiments, the anti-LAM antibody or anti-PIM6/LAM
antibody is a humanized monoclonal anti-LAM antibody or
anti-PIM6/LAM antibody, respectively. In some embodiments, the
anti-LAM antibody binds to Ara4 and Ara6 structures.
In some embodiments, the LAM epitope is an uncapped arabinose
chain. In some embodiments the LAM epitope is an uncapped or single
mannose capped arabinose chain, with or without a terminal MTX
substitution.
In some embodiments, the LAM epitope is a mannose-capped Ara4
structure and a mannose-capped Ara6 structure. In other
embodiments, the anti-LAM antibody specifically binds to an
.alpha.(1.quadrature.2)-linked dimannose structure, which may be
joined either to an Ara4/Ara6 structure, or to a polymannose
structure (FIG. 8). In some embodiments, the PIM6 epitope includes
at least one polymannose structure also present in mycobacterial
lipomannan (LM). In some embodiments the anti-PIM6/LAM antibody
specifically binds to a PIM6 epitope that includes at least one
polymannose structure in the PIM6 mannan domain. In some
embodiments, the LAM epitope includes at least one methylthioxylose
(MTX) or methylsylfinylxylofuranosyl (MSX) substitution. In some
embodiments, the LAM epitope includes at least one
phosphatidyl-myo-inositol substitution (PILAM). In some
embodiments, the LAM epitope is an arabinose chain capped with at
least one mannose, i.e. mannosylated Man-LAM epitope. In further
embodiments, the capped arabinose chain includes Ara4 and/or Ara6
structures. In some embodiments, the Man-LAM epitope includes
mono-mannose substituted arabinose chains, di-mannose substituted
arabinose chains, tri-mannose substituted arabinose chains, or
combinations thereof. In some embodiments, the Man-LAM epitope
includes di-mannose or tri-mannose capped Ara4 and/or Ara6
structures. In some embodiments, the Man-LAM epitope is di-mannose
capped Ara6. In some embodiments, the anti-LAM antibody or
anti-PIM6/LAM antibody includes an IgG antibody. In further
embodiments, the IgG anti-LAM antibody or anti-PIM6/LAM antibody
includes a subclass of IgG1, IgG2 or IgG3. In some embodiments, the
anti-LAM antibody or anti-PIM6/LAM antibody is not an IgG antibody.
In other embodiments, the anti-LAM antibody or anti-PIM6/LAM
antibody includes an IgA antibody. In other embodiments, the
anti-LAM antibody or anti-PIM6/LAM antibody includes an IgM
antibody. In some embodiments, the anti-LAM antibody or
anti-PIM6/LAM antibody includes a second isotype that has been
switched from the isotype originally isolated. In some embodiments,
the anti-LAM antibody or anti-PIM6/LAM antibody includes a
recombinant antibody. In some embodiments, the recombinant antibody
includes a multivalent IgM antibody. In further embodiments, the
recombinant antibody includes a pentavalent IgM antibody. In other
embodiments, the recombinant antibody includes an ScFv-IgG
antibody, in which a single chain Fv fragment of one antibody is
joined to the N-terminus of the heavy chain of that or a different
anti-LAM mAb. In further embodiments, the recombinant antibody
includes a multivalent ScFv-IgG antibody. In further embodiments,
the recombinant antibody includes a homologous tetravalent ScFv-IgG
antibody, in which the scFv domains were derived from the variable
regions of the IgG present in the construct. In yet further
embodiments, the recombinant antibody includes a heterologous
tetrameric scFv-IgG antibody in which the scFv regions were derived
from a different anti-LAM antibody or anti-PIM6/LAM antibody as the
IgG region included. In some embodiments, the scFv domain includes
a leader sequence joined to the variable heavy (VH) region of
second anti-LAM antibody or anti-PIM6/LAM antibody which is joined
to the variable light (VL) domain of said anti-LAM antibody or
anti-PIM6/LAM antibody. In other embodiments, the scFv domain
includes a leader sequence joined to the variable light chain
region of a first anti-LAM antibody or anti-PIM6/LAM antibody which
is joined to the variable heavy (VH) region of a second anti-LAM
antibody or anti-PIM6/LAM antibody. In some embodiments, the
anti-LAM antibody is an isolated anti-LAM antibody that
specifically binds to a LAM epitope (e.g., one of Ara4 and Ara6 or
combinations thereof, an .alpha.(1.fwdarw.2)-linked dimannose
structure, which may be joined either to an Ara4/Ara6 structure, or
to a polymannose structure). In some embodiments, the anti-LAM
antibody does not compete with CS-35 and FIND25. In some
embodiments, the anti-PIM6/LAM antibody is an isolated
anti-PIM6/LAM antibody that specifically binds to at least one
polymannose structure in mycobacterial lipomannan (LM).
In some embodiments, the anti-LAM antibody or anti-PIM6/LAM
antibody includes a flexible linker. In some embodiments, the
flexible linker joins the corresponding heavy and light chain
domains into a single chain molecule. In some embodiments, the
flexible linker connects an immunoglobulin light chain (IgVL) to an
immunoglobulin heavy chain (IgVH). In further embodiments, the
flexible linker is comprised of the formula (GGSGG)n (SEQ ID
NO:19), wherein n is any positive integer between 1 and 200 and any
ranges in between, e.g. 1 to 5, 1 to 10, 1 to 15, 1 to 25, 1 to 50,
5 to 10, 5 to 25, 10 to 25, 10 to 50, 1 to 100, 1 to 150, and all
intervening ranges.
In some embodiments, the anti-LAM antibody (e.g., P30B9, A194-01)
has at least one (e.g., one, two, three) complimentarity
determining region (CDR) (e.g. CDR1, CDR2, CDR3). In some
embodiments, the variable light region of CDR1 consists essentially
of SEQ ID NO: 1 or antigenic fragments thereof. In other
embodiments, the variable light region of CDR1 region consists
essentially of SEQ ID NO: 7 or antigenic fragments thereof. In
other embodiments, the variable light region of CDR1 region
consists essentially of SEQ ID NO: 13 or antigenic fragments
thereof. In some embodiments, the variable heavy region of CDR1
consists essentially of SEQ ID NO: 4 or antigenic fragments
thereof. In other embodiments, the variable heavy region of CDR1
region consists essentially of SEQ ID NO: 10 or antigenic fragments
thereof. In other embodiments, the variable heavy region of CDR1
region consists essentially of SEQ ID NO: 16 or antigenic fragments
thereof.
In some embodiments, the variable light region of CDR2 consists
essentially of SEQ ID NO: 2 or antigenic fragments thereof. In
other embodiments, the variable light region of CDR2 consists
essentially of SEQ ID NO: 8 or antigenic fragments thereof. In
other embodiments, the variable light region of CDR2 consists
essentially of SEQ ID NO: 14 or antigenic fragments thereof. In
some embodiments, the variable heavy region of CDR2 consists
essentially of SEQ ID NO: 5 or antigenic fragments thereof. In
other embodiments, the variable heavy region of CDR2 region
consists essentially of SEQ ID NO: 11 or antigenic fragments
thereof. In other embodiments, the variable heavy region of CDR2
region consists essentially of SEQ ID NO: 17 or antigenic fragments
thereof.
In some embodiments, the variable light region of CDR3 consists
essentially of SEQ ID NO: 3 or antigenic fragments thereof. In
other embodiments, the variable light region of CDR3 consists
essentially of SEQ ID NO: 9 or antigenic fragments thereof. In
other embodiments, the variable light region of CDR3 consists
essentially of SEQ ID NO: 15 or antigenic fragments thereof. In
some embodiments, the variable heavy region of CDR3 consists
essentially of SEQ ID NO: 6 or antigenic fragments thereof. In
other embodiments, the variable heavy region of CDR3 region
consists essentially of SEQ ID NO: 12 or antigenic fragments
thereof. In other embodiments, the variable heavy region of CDR3
region consists essentially of SEQ ID NO: 18 or antigenic fragments
thereof.
In some embodiments, the anti-PIM6/LAM antibody (e.g., P95C1) has
at least one (e.g., one, two, three) CDR (e.g., CDR1, CDR2, CDR3).
In some embodiments, the variable light region of CDR1 consists
essentially of SEQ ID NO: 13 or antigenic fragments thereof. In
some embodiments, the variable heavy region of CDR1 consists
essentially of SEQ ID NO: 16 or antigenic fragments thereof. In
some embodiments, the variable light region of CDR2 consists
essentially of SEQ ID NO: 14 or antigenic fragments thereof. In
some embodiments, the variable heavy region of CDR2 consists
essentially of SEQ ID NO: 17 or antigenic fragments thereof. In
some embodiments, the variable light region of CDR3 consists
essentially of SEQ ID NO: 15 or antigenic fragments thereof. In
some embodiments, the variable heavy region of CDR3 consists
essentially of SEQ ID NO: 18 or antigenic fragments thereof.
B. Diagnostic Kits and Methods
In some embodiments, the present invention provides kits for
detecting the presence of LAM and/or PIM6 in biological fluids of a
human subject. In some embodiments the components of this assays
are assembled in a lateral flow device (see World Health
Organization 2015, The use of lateral flow urine lipoarabinomannan
assay (LF-LAM) for the diagnosis and screening of active
tuberculosis in people living with HIV). In some embodiments, the
kits include a first anti-LAM (e.g., A194-01, P30B9) or
anti-PIM6/LAM (e.g., P95C1) capture antibody, a second anti-LAM or
anti-PIM6/LAM detector (detection) antibody labeled with a reporter
molecule, a support for which the first anti-LAM or anti-PIM6/LAM
antibody is bound to, and a buffer. In some embodiments, at least
one of the first anti-LAM or anti-PIM6/LAM antibody and the second
anti-LAM or anti-PIM6/LAM antibody is a human monoclonal anti-LAM
antibody that binds specifically to one of Ara4 and Ara6 or
combinations thereof, or a human monoclonal anti-PIM6/LAM antibody
that binds specifically to the mannan domain of LAM (and lipomannan
(LM)). In some embodiments, the first anti-LAM antibody and the
second anti-LAM antibody bind to the same LAM epitopes which are
present in multiple copies on a single LAM molecule. In other
embodiments, the first anti-LAM antibody and the second anti-LAM
antibody bind to different epitopes present on a single LAM
molecule. The LAM and PIM6 epitopes may be any of the LAM and PIM6
epitopes described herein. In other embodiments, a third detector
(detection) antibody is included which binds to a non-competing
site of the second antibody. In some embodiments, the first
antibody and the second antibody are of the same isotype. In other
embodiments, the first antibody and the second antibody are
different isotypes. In some embodiments of a capture assay, only
either the capture antibody or the detection antibody is an
anti-LAM antibody (e.g., A194-01, P30B9) or an anti-PIM6/LAM
antibody (e.g., P95C1) as described herein.
The antibodies described herein can be used for additional
detection and diagnostic applications. For example, in one
diagnostic assay, one or more of the antibodies described herein
(e.g., A194-01, P30B9, P95C1) can be used to stain tissues obtained
from patients to detect the presence of LAM in lesions suspected of
containing TB or TB-infected cells (e.g., granulomas). This can be
done, for example, with a single antibody as described herein
(e.g., A194-01, P30B9, P95C1) that is conjugated with a label that
allows sensitive detection. In such a method or assay, detection by
P95C1 of PIM6 or related molecules can be achieved in infected
tissues. In another example, P95C1 can be used in a PIM6
competition assay, in which the capture of a labeled form of PIM6
by immobilized P95C1 is competed by soluble PIM6 present in a
biological fluid (e.g., blood or urine) of a suspect. In the
absence of soluble PIM6, this would result in the capture of a
signal, which would be competed by the presence of soluble PIM6
(see World Health Organization, 2015 Policy Guidance--The use of
lateral flow urine lipoarabinomannan assay (LF-LAM) for the
diagnosis and screening of active tuberculosis in people living
with HIV).
In some embodiments, the present invention provides kits for
distinguishing between a pathogenic member of the Mycobacterium
tuberculosis-complex and a nonpathogenic member of the
Mycobacterium tuberculosis-complex. In some embodiments, the
anti-LAM antibody is a human monoclonal anti-LAM antibody that
binds specifically to one of Ara4 and Ara6 structure with or
without a Man or MTX-Man substitution or combinations thereof, or
anti-PIM6/LAM antibody that specifically binds at least one
polymannose structure in PIM6 or in the LAM mannan domain. In some
embodiments, the anti-LAM antibody specifically binds to a Man-LAM
epitope including di-mannose substituted side chains, tri-mannose
substituted side chains, or combinations thereon. In further
embodiments, the anti-LAM antibody specifically binds to Man-LAM
epitopes includes di-mannose or tri-mannose capped Ara4 and/or Ara6
structures. In yet further embodiments, the anti-LAM antibody
specifically binds to di-mannose capped Ara6 structures.
In some embodiments, the present invention provides methods for
diagnosing an active tuberculosis infection in an individual. In
some embodiments the anti-LAM or anti PIM6/LAM antibody can be
modified with a sensitive tag and used to identify mycobacterial
PIM6 or LAM-related material in a tissue sample, as a diagnostic
for TB infection and localization. In some embodiments, the method
involves the capture of soluble LAM, and includes the steps of (a)
obtaining a sample from an individual that includes LAM; (b)
treating the sample to isolate or expose said LAM, (c) capturing
said isolated or exposed LAM with a first anti-LAM antibody that
binds to a first epitope on said LAM; (d) contacting said isolated
or exposed LAM with a second anti-LAM antibody, wherein said second
anti-LAM antibody binds to a second epitope on said LAM; (e)
detecting the binding of at least one of said first anti-LAM
antibody and said second anti-LAM antibody to said LAM; and (f)
diagnosing said patient as having an active tuberculosis infection,
wherein said presence of binding of at least one of said first
anti-LAM antibody and said second anti-LAM antibody to said LAM
indicates an active tuberculosis infection. In some embodiments, at
least one of the first anti-LAM antibody and the second anti-LAM
antibody is a human monoclonal anti-LAM antibody that binds
specifically to one of Ara4 and Ara6 or combinations thereof. In
some embodiments, at least one of the first and second antibodies
is a human monoclonal anti-PIM6/LAM antibody that specifically
binds to at least one polymannose structure in the LAM mannan
domain. In further embodiments, the first antibody and the second
antibody are different isotypes. In some embodiments, at least one
of the first antibody and the second antibody are recombinant
antibodies. In other embodiments, neither the first antibody nor
the second antibody are recombinant antibodies. In yet other
embodiments, both the first antibody and the second antibody are
recombinant antibodies.
In some embodiments, the present invention provides methods of
quantifying the amount of LAM and/or PIM6 present in a sample. In
some embodiments, the method includes the steps of (a) obtaining a
sample that includes LAM and/or PIM6; (b) contacting said sample
with an anti-LAM antibody and/or an anti-PIM6 antibody; (c)
detecting the presence of specific binding of the anti-LAM antibody
to said LAM and/or the binding of the anti-PIM6/LAM antibody to
said LAM or said PIM6; and (d) quantifying the amount of LAM or
PIM6 in said sample. In some embodiments, the anti-LAM antibody is
a human monoclonal anti-LAM antibody that binds specifically to one
of Ara4 and Ara6 or combinations thereof. In some embodiments, the
anti-PIM6/LAM antibody is a human monoclonal anti-PIM6/LAM antibody
that binds specifically to at least one polymannose structure in
the PIM6 mannan domain (e.g., to at least one polymannose structure
in mycobacterial lipomannan (LM)). In some embodiments, quantifying
said amount of LAM and/or PIM6 is achieved by comparing the signal
intensity to that of a serially diluted control sample having a
known concentration of LAM and/or PIM6.
In some embodiments the present invention provides methods for
diagnosing an individual as being infected with Mycobacterium
tuberculosis. In some embodiments, the method includes the steps of
(a) obtaining a sample that includes LAM or PIM6; (b) contacting
said sample with an anti-LAM antibody and/or an anti-PIM6 antibody,
wherein the anti-LAM antibody binds specifically to a LAM epitope
including Man-LAM having at least one at least one
5-deoxy-5-methylthiopentofuranosyl (MTX) substitution, and the
anti-PIM6/LAM antibody binds specifically to an epitope including
at least one polymannose structure in the LAM mannan domain, and
(c) detecting the presence of specific binding of the anti-LAM
antibody to said Man-LAM and/or the presence of specific binding of
the anti-PIM6/LAM antibody to said PIM6. In some embodiments, the
anti-LAM antibody is a human monoclonal anti-LAM antibody that
binds specifically to one of Ara4 and Ara6 or combinations thereof.
In some embodiments, the anti-PIM6/LAM antibody is a human
monoclonal anti-PIM6/LAM antibody (e.g., P95C1) that binds
specifically to at least one polymannose structure in the PIM6
mannan domain.
In some embodiments the method includes an amplification step that
increases the sensitivity of the detection method. Examples involve
the generation of additional target sites by the use of Tyramide
Signal Amplification kit (Perkin-Elmer) or the amplification of the
initial signal by the use of the ELISA Amplification System (Thermo
Fisher).
In some embodiments, the present invention provides methods of
differentiating between a pathogenic member of the Mycobacterium
tuberculosis-complex and a nonpathogenic member of the
Mycobacterium tuberculosis-complex. In some embodiments, the method
includes the steps of (a) obtaining a sample that comprises LAM
and/or PIM6; (b) contacting said sample with an anti-LAM antibody
that binds specifically to a Man-LAM epitope that includes
di-mannose substituted side chains, tri-mannose substituted side
chains, or combinations thereof, or with an anti-PIM6/LAM antibody
that binds specifically to at least one polymannose structure in
the PIM6 mannan domain; and (c) detecting the presence of specific
binding of the anti-LAM antibody to said Man-LAM, or the presence
of the specific binding of the anti-PIM6/LAM antibody to said at
least one polymannose structure in the PIM6 mannan domain, wherein
the presence of said specific binding indicates the presence of a
pathogenic member of the Mycobacterium tuberculosis-complex. In
some embodiments, the anti-LAM antibody is a human monoclonal
anti-LAM antibody that binds specifically to one of Ara4 and Ara6
or combinations thereof. In further embodiments, the Man-LAM
epitope includes di-mannose or tri-mannose capped Ara4 and/or Ara6
structures. In yet further embodiments, the Man-LAM epitope is
di-mannose capped Ara6. In some embodiments, the anti-PIM6/LAM
antibody is a human monoclonal anti-PIM6/LAM antibody that binds
specifically to at least one polymannose structure in the PIM6
mannan domain.
C. Therapeutic Compositions, Methods, Vaccines, and Vectors
In some embodiments, the present invention provides methods for
treating infection by a virulent member of the Mycobacterium
tuberculosis-complex in an individual. In some embodiments, the
method includes administering a therapeutically effective amount of
at least one anti-LAM antibody or anti-PIM6/LAM antibody to an
individual exposed to infectious M.tb. In further embodiments, the
method includes administration of at least one antibiotic. In some
embodiments, the TB infection is active. In other embodiments, the
TB infection is latent. In some embodiments, the infection is with
a multiple-drug resistant (MDR) strain of tuberculosis. In other
embodiments, the infection is with an extensively-drug resistant
(XDR) strain of tuberculosis.
In some embodiments, the present invention provides a combination
therapy for treating infection by a virulent member of the
Mycobacterium tuberculosis-complex in an individual. In some
embodiments, the method includes administering a therapeutically
effective amount of a first anti-LAM antibody that specifically
binds to a first LAM epitope including at least one of
unsubstituted LAM, mono-mannosylated Man-LAM, MSX-substituted LAM,
and combinations thereof or a first anti-PIM6/LAM antibody that
specifically binds to at least one polymannose structure in the
PIM6 and LAM mannan domain; and administering a therapeutically
effective amount of a second anti-LAM antibody that specifically
binds to a second LAM epitope including at least one of di-mannose
substituted Man-LAM, tri-mannose substituted Man-LAM, and
combinations thereof. In some embodiments, the first antibody and
the second antibody are administered simultaneously (e.g., in a
single composition, or in two compositions administered at the same
time). In other embodiments, the first antibody and the second
antibody are administered at different time points. In some
embodiments, at least one of the first anti-LAM antibody and the
second anti-LAM antibody is a human monoclonal anti-LAM antibody
that binds specifically to one of Ara4 and Ara6 or combinations
thereof. In some embodiments, the anti-PIM6/LAM antibody is a human
monoclonal anti-PIM6 antibody that binds specifically to at least
one polymannose structure in PIM6 and/or in the PIM6 crossreactive
mannan domain of LAM. In some embodiments, the first anti-LAM
antibody and the second anti-LAM antibody, or the anti-PIM6/LAM
antibody are of different isotypes. In some embodiments, at least
one of the first anti-LAM antibody and the second anti-LAM
antibody, and the anti-PIM6/LAM antibody are recombinant
antibodies. In other embodiments, neither the first anti-LAM
antibody nor the second anti-LAM antibody, nor the anti-PIM6/LAM
antibody, are recombinant antibodies. In yet other embodiments,
both the first anti-LAM antibody and the second anti-LAM antibody,
or the anti-PIM6/LAM antibody, are recombinant antibodies. In
further embodiments, the method includes administration of at least
one antibiotic. In such embodiments, the at least one antibiotic
can be administered (e.g., co-administered) simultaneously with the
first and second antibodies, or the at least one antibiotic can be
administered at a time point different from the time point of
administration of the first and second antibodies. In some
embodiments, the infection is active. In other embodiments, the
infection is latent. In some embodiments, the infection is a
multiple-drug resistant (MDR) tuberculosis infection. In other
embodiments, the infection is an extensively-drug resistant (XDR)
tuberculosis infection.
In some embodiments, the present invention provides vaccines or
pharmaceutical compositions for preventing infection by a virulent
member of the Mycobacterium tuberculosis-complex. In some
embodiments, the invention provides a method of stimulating a host
immune response in a patient including administering to said
patient a therapeutically effective amount of a LAM antigen and/or
a PIM6 antigen. In some embodiments these antigens are conjugated
to immunogenic protein carriers, and/or are co-administered with an
adjuvant that potently stimulates an immune response to glycolipid
antigens. In some embodiments, the vaccine or pharmaceutical
composition induces an anti-LAM antibody that specifically binds to
a Man-LAM epitope, and/or an anti-PIM6/LAM antibody that
specifically binds to at least one polymannose structure in the
PIM6 mannan domain. In further embodiments, the Man-LAM epitope
present in the vaccine or pharmaceutical compositions includes
di-mannose or tri-mannose capped Ara4 and/or Ara6 structures. In
yet further embodiments, the Man-LAM epitope is di-mannose capped
Ara6. In some embodiments, the Man-LAM epitope has at least one MTX
substitution. In some embodiments, the anti-LAM antibody and/or
anti-PIM6/LAM antibody is an IgM antibody. In other embodiments,
the anti-LAM antibody and/or anti-PIM6/LAM antibody is a
recombinant antibody.
In some embodiments, the present invention provides a method of
preventing infection by a virulent member of the Mycobacterium
tuberculosis-complex in an individual by passive administration of
a protective antibody. In some embodiments, the anti-LAM antibody
is a human monoclonal anti-LAM antibody that binds specifically to
one of Ara4 and Ara6 or combinations thereof. In some embodiments,
the anti-PIM6/LAM antibody is a human monoclonal anti-PIM6/LAM
antibody that binds specifically to at least one polymannose
structure in the PIM6 and LAM mannan domain. In some embodiments,
the method includes administering to an individual a
therapeutically effective amount of an anti-LAM antibody that
specifically binds to a Man-LAM epitope, and/or an anti-PIM6
antibody that specifically binds to a PIM6 epitope (e.g., an
epitope shared by PIM6 and LAM). In further embodiments, the
targeted ManLAM epitope includes di-mannose or tri-mannose capped
Ara4 and/or Ara6 structures. In yet further embodiments, the ManLAM
epitope is di-mannose capped Ara6. In some embodiments, the ManLAM
eptiope has at least one MTX substitution. In some embodiments, the
anti-LAM antibody or anti-PIM6/LAM antibody is an IgM antibody. In
other embodiments, the anti-LAM antibody or anti-PIM6/LAM antibody
is a recombinant antibody.
In some embodiments, the present invention provides passive
administration of a protective antibody via recombinant vectors. In
some embodiment, the recombinant vectors include a first nucleic
acid encoding for an IgVL of an anti-LAM antibody and a second
nucleic acid encoding an IgVH of an anti-LAM antibody, wherein each
of the nucleic acids is operably linked to a promoter region. In
some embodiments, at least one of the IgVL and IgVH is derived from
a human monoclonal anti-LAM antibody that binds specifically to one
of Ara4 and Ara6 or combinations thereof. In other embodiment, the
recombinant vectors include a first nucleic acid encoding for an
IgVL of an anti-PIM6/LAM antibody and a second nucleic acid
encoding an IgVH of an anti-PIM6/LAM antibody, wherein each of the
nucleic acids is operably linked to a promoter region. In some
embodiments, the recombinant vectors include additional
transcriptional regulation elements. In some embodiments, at least
one of the first nucleic acid sequence and the second nucleic acid
sequence are organized in an operon. In some embodiments, at least
one of the first nucleic acid sequence and the second nucleic acid
sequence are organized in an expression cassette. In some
embodiments, the first nucleic acid sequence and the second nucleic
acid sequence are organized in a single expression cassette. In
some embodiments, the first nucleic acid and the second nucleic
acid are located in the same cloning vector. In other embodiments,
the first nucleic acid and the second nucleic acid are located in
different cloning vectors. In some embodiments, expression of the
first nucleic acid and the second nucleic acid may be concomitant.
In other embodiments, expression of the first nucleic acid and the
second nucleic acid is separably inducible. In some embodiments,
expression of the first nucleic acid may be temporally separate
from expression of the second nucleic acid. In some embodiments,
the recombinant vector is a plasmid. In other embodiments, the
recombinant vector is a non-replicated virus. In further
embodiments, the recombinant vector is an adeno-associated
virus.
In some embodiments, the present invention provides for a method of
treating infection by a virulent member of the Mycobacterium
tuberculosis-complex in an individual. In some embodiments, the
method includes administering to an individual a first nucleic acid
coding for an IgVH of an anti-LAM antibody, and a second nucleic
acid coding for an IgVL of an anti-LAM antibody, wherein each of
the nucleic acids is operably linked to a promoter region. In other
embodiments, the method includes administering to an individual a
first nucleic acid coding for an IgVH of an anti-PIM6/LAM antibody,
and a second nucleic acid coding for an IgVL of an anti-PIM6/LAM
antibody, wherein each of the nucleic acids is operably linked to a
promoter region. In some embodiments, at least one of the IgVL and
IgVH is derived from a human monoclonal anti-LAM antibody that
binds specifically to one of Ara4 and Ara6 or combinations thereof,
or from a human monoclonal anti-PIM6/LAM antibody that binds
specifically to at least one polymannose structure in the PIM6
mannan domain. In some embodiments, the first nucleic acid and the
second nucleic acid are located in the same cloning vector. In
other embodiments, the first nucleic acid and the second nucleic
acid are located in different cloning vectors. In some embodiments,
the recombinant vector is a plasmid. In other embodiments, the
recombinant vector is a non-replicated virus. In further
embodiments, the recombinant vector is an adeno-associated
virus.
Additional embodiments, features and advantages will be readily
apparent to one of skill in the art based on the disclosure
provided herein. Other features will become more apparent to
persons having ordinary skill in the art to which the package
pertains and from the following description and claims. Although
antibodies, compositions, kits and methods similar or equivalent to
those described herein can be used in the practice or testing of
the present invention, suitable antibodies, compositions, kits and
methods are described below. All publications, patent applications,
and patents mentioned herein are incorporated by reference in their
entirety. In the case of conflict, the present specification,
including definitions, will control. The particular embodiments
discussed below are illustrative only and not intended to be
limiting.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A--Model of IgG form of A194-01 and fragments thereof used in
binding competition assays. These included monovalent scFv and Fab
structures, and divalent scFv dimer and natural IgG.
1B--Competition curves showing that the monovalent forms of A194-01
competed less effectively than the divalent forms. 1C--Structure of
higher-valent form of A194-01. This represents a homologous
tetravalent A194-01 scFv-IgG, which contains an A194-01 scFv domain
joined to the N-terminus of each of the normal heavy chains.
FIG. 2A--Binding activity of P30B9 IgG and IgM forms, and IgM in
which the 6 amino acid insert in the VH region was deleted, or the
9 somatic mutatic mutations in the VH region were reverted to the
nearest germ-line sequence, to ManLAM derived from Mycobacterium
tuberculosis. The 6 amino acid insert contributed to a greater
extent than the 9 somatic acid mutations to reactivity. FIGS. 2B
and 2C compare the reactivity of the P30B9 IgM and IgG forms and
the mutation with the 6 amino acid deletion in the heavy chain
against ManLAM from Mycobacterium tuberculosis (B) and PILAM from
Mycobacterium smegmatis (C). The IgM form, but not the IgG form,
reacted specifically with ManLAM derived from Mycobacterium
tuberculosis (2B) but not PILAM (2C), and the reactivity of the
.DELTA.6 amino acid mutant was highly reduced for ManLAM and
negative for PILAM.
FIG. 3--Comparing the reactivity of 2 human mAbs and 4 mouse mAbs
vs PILAM in the left panel and vs. ManLAM isolated from the H37Rv
strain of Mycobacterium tuberculosis in the right panel. Curves
were plotted using the molar concentrations of the antibodies, to
control for the different molecular weights of these reagents
FIG. 4A--Structures of 25 synthetic oligosaccharides representing
microbial glycan structures related to motifs present in LAM. These
structures were coupled to BSA carrier protein and used to probe
epitope specificity. 4B--Binding profiles for six LAM-specific
monoclonal antibodies against panel of 25 synthetic
oligosaccharides. Binding results are shown for three
concentrations, and the relative affinities of the antibodies to
these antigens is indicated by the titration pattern.
FIG. 5--Left hand panel-structure of IgA1 (A), IgA2 (B) and dimeric
IgA1-J dimeric complex (C). Right hand panel-SDS-PAGE gel of
purified P30B9 IgA1, IgA2 and IgA3 proteins, both before and after
reduction with DTT. P30B9 IgA3 was later revealed to be an artifact
of PCR with a longer hinge region.
FIG. 6--Binding curves of different isotypes of P30B9 to ManLAM
showing greatest activity for IgM form followed by IgA forms, with
no reactivity for the IgG form.
FIG. 7--Comparison of efficiency of biotinylated monoclonal
antibody probes at detecting soluble ManLAM in CS-35 capture assay,
in which the indicated concentration of ManLAM was captured by
CS-35 and detected by the indicated mAbs labeled with biotin.
FIG. 8--Binding curves of P30B9 to various mannose-capped Ara4
structures, or to tetra- and penta-mannose structures. Preferential
binding was seen for structures 3 (dimannose-Ara4) and 59, which
contained the related .alpha.-Manp(1.quadrature.2)-Manp
linkage.
FIG. 9--Titration of monoclonal anti-LAM antibodies against various
uncapped LAM-related glycoconjugates to determine structural
requirements for reactivity. 9A-Analysis of the importance of the
Ara-.alpha.(1.quadrature.5)-Ara linkage at the penultimate position
from the non-reducing end of the Ara4 sequence. 9B-- Analysis of
the dependence of the Ara-.beta.(1.quadrature.2)-Ara linkage at the
terminal position of the Ara4 sequence.
FIG. 10 Binding curves of A194-01 IgG and three murine anti-LAM
antibodies against various Arab-containing glycoconjugates, showing
the effect of different capping motifs on antibody reactivity.
FIG. 11 Binding competition studies to measure the ability of
individual anti-LAM antibodies to compete for binding of a probe
antibody to the ManLAM antigen. Antibodies were biotinylated when
tested against antibodies from the same species. Note inability of
A194-01 to compete for biotinylated P30B9.
FIG. 12--Competition of binding of anti-LAM monoclonal antibodies
to LAM derived from Mycobacterium tuberculosis (ManLAM) and LAM
derived from Mycobacterium smegmatis (PILAM). Efficient competition
between FIND25 and P30B9 for ManLAM is consistent with predominance
of dimannose-substituted Arab, while lack of competition of these
two mAbs by A194 is consistent with its poor reactivity with
dimannose capped structures. The efficient competition of A194 for
FIND25 vs PILAM is consistent with the absence of dimannose capping
in this structure.
FIG. 13--Binding competition of biotinylated probe monoclonal
antibodies with excess unmodified antibodies against natural LAM
antigens and selected glycoconjugates. 13A--Competition of binding
of biotinylated A194-01 IgG, CS-35 and FIND25 to MAnLAM by four
mAbs; 13B-- Competition of binding of FIND25 to both ManLAM and
PILAM by three mAbs; 13C-- Competition of binding of P30B9 IgM to
MAnLAM and two synthetic glycoconjugate antigens by four mAbs.
FIG. 14--Engineered variants and/or derivatives of A194-01 react
with a broader range of glycoconjugates, including di- and
tri-mannose substituted forms poorly recognized by the IgG isotype
of A194-01.
FIG. 15--Differential competition of A194-01 IgG and engineered
variants and/or derivatives of A194-01 for binding of FIND25 and
P30B9 IgM to ManLAM. Although A194 IgG doesn't compete with P30B9
or FIND25 against ManLAM, the multimeric forms do compete,
consistent with the broader epitope specificity of these forms. As
shown above, A194 IgG does compete well with FIND25 for PILAM.
FIG. 16--Comparison of analysis of the effect of mannose-capping on
the reactivity of CS-40, A194-01 and P30B9 mAbs. Antibody binding
specificities were measured by ELISA against specific glyconjugates
containing different mannose substitutions. The antibody titrations
are shown in 16A and the structures of the mannose-containing
glycan antigens is shown in 16B.
FIG. 17-17A. Homologous scFv-IgG. In this example, both the IgG and
scFv domains are derived from the same antibody. This results in an
increased valency (tertavalent vs. divalent) but does not directly
modify the target specificity. 17B. Heterologous scFv-IgG. In
addition to the increase in valency, there is also a broadened
specificity introduced, which may allow recognition of distinct
epitopes in a single antigen molecule. 17C. Heterologous scFv-IgM.
In this formulation a distinct scFv is combined with an IgM
construct. One example would be joining of the A194-01 scFv with
the P30B9 IgM. In addition to the increase in valency, this would
introduce an additional epitope specificity, which may allow
multivalent recognition of distinct epitopes that may not be
recognized by the homologous scFv-IgM, and lead to increased
affinities.
FIG. 18A-18C--Mapping of epitopes recognized by new mAbs. The
epitope specificity of P95C1 was compared to that of two previously
described mAbs, A194-01 and P30B9, and two new mAbs, P61H5 and
P83A8, that recognize epitopes related to those two previously
described mAbs. 18A. Reactivity of LAM-specific mAbs for LAM
precursor molecules. P30B9 and P61H5 were specific for ManLAM over
PILAM, while A194-01, P83A8 and P95C1 recognized both forms of LAM.
P95C1 also bound efficiently with LM and PIM6. The weak reactivity
of the other mAbs for LM and PIM6 is die at least in part, to
contamination of these materials by ManLAM. 18B. Reactivity of
synthetic LAM-derived glycoconjugates. 18C. In contrast to
previously known mAbs, P95C1 was the only antibody that did not
recognize any of the polyarabinose structures, but reacted
specifically with two poly-mannose structures, YB-BSA-05 and
YB-BSA-11, that resembled structures present in PIM6 and in the
mannan domains at the base of LM and LAM.
FIG. 19--Effect of isotype switching on binding of P95C1 and P30B9
to ManLAM and PI-LAM. For P95C1, IgM, IgA and IgG isotypes all have
comparable binding activity for both ManLAM and PILAM, unlike P30B9
which react only with ManLAM and only in IgM and IgA forms but not
as IgG.
FIG. 20(A)-20(B)--Western blot analysis of crossreactivity of P95C1
with LAM and additional M.tb glycolipids. 20(A) Purified LAM
associated glycolipids were separated on 12% SDS-PAGE gel followed
by oxidation and staining of sugar molecules with periodic
acid-Schiff stain, to reveal material containing reactive glycans.
20(B) Parallel blots were probed with mAbs A194 IgG1, P30B9 IgM,
and P95C1 IgM followed by alkaline phosphatase conjugated anti
human IgG and IgM secondary antibodies and treatment with bcip/nbt
color development substrate. A194-01 crossreacts with ManLAM from
M.tb and PILAM from M.smeg. P30B9 is specific for M.tb ManLAM.
P95C1 recognizes both species of LAM, as well as LM and PIM6
isolated from M.tb. Weak staining by A194-01 of bands in LM and
PIM6 that co-migrate with LAM is apparently due to minor
contamination of these samples with LAM.
FIG. 21--Alignments of amino acid sequences for A194 heavy chain
and light chain variable regions sequences and their comparison
with their closest germline sequences. In the top alignment, from
the top, the first amino acid sequence (A194-VH) is an A194 heavy
chain variable region sequence without the CDR3 sequence (SEQ ID
NO:23). The heavy chain variable region sequence without CDR3 is
SEQ ID NO:21. In the top alignment, the second amino acid sequence
(germline Homsap IGHV3-20*01) is SEQ ID NO:22. In the top
alignment, the third amino acid sequence is the CDR3 of a A194
heavy chain variable region and is SEQ ID NO:23. In the bottom
alignment, from the top, the first amino acid sequence (A-194-Vk)
is an A194 light chain variable region without the CDR3 sequence
(SEQ ID NO: 26). The light chain variable region sequence without
CDR3 is SEQ ID NO:24. In the bottom alignment, the second amino
acid sequence (germline Homsap IGKV3-15*01) is SEQ ID NO:25. In the
bottom alignment, the third sequence is the CDR3 of a A194 light
chain variable region and is SEQ ID NO:26.
FIG. 22--Amino acid sequences for P30B9-IgM heavy chain and light
chain variable region sequences and their comparisons with their
closest germlines. In the top alignment, from the top, the first
amino acid sequence (P30B9-Vh) is a P30B9-IgM heavy chain variable
region sequence without the CDR3 sequence (SEQ ID NO:29). The heavy
chain variable region sequence without CDR3 is SEQ ID NO: 27. The
second amino acid sequence (Homsap IGHV4-34*01 F) is SEQ ID NO:28.
The third amino acid sequence is of a P30B9-IgM heavy chain
variable region and is SEQ ID NO:29. In the bottom alignment, from
the top, the first amino acid sequence (P30B9-Vk) is a P30B9 light
chain variable region without the CDR3 sequence (SEQ ID NO:32). The
light chain variable region sequence without CDR3 is SEQ ID NO:30.
In the bottom alignment, the second amino acid sequence (germline
Homsap IGKV1-5*03) is SEQ ID NO:31. In the bottom alignment, the
third sequence is the CDR3 of a P30B9 light chain variable region
and is SEQ ID NO:32.
FIG. 23--Alignments of amino acid sequences for P95C1-IgM heavy
chain and light chain variable regions sequences and their
comparison with their closest germline sequences. In the top
alignment, from the top, the first amino acid sequence (P95C1-VH)
is an P95C1 heavy chain variable region sequence without the CDR3
sequence (SEQ ID NO:18). The heavy chain variable region sequence
without CDR3 is SEQ ID NO:33. In the top alignment, the second
amino acid sequence (germline Homsap IGHV4-4*02) is SEQ ID NO:34.
In the top alignment, the third amino acid sequence is the CDR3 of
a P95C1-gM heavy chain variable region and is SEQ ID NO:18. In the
bottom alignment, from the top, the first amino acid sequence
(P95C1-Vk) is a P95C1 light chain variable region without the CDR3
sequence (SEQ ID NO:15). The light chain variable region sequence
without CDR3 is SEQ ID NO:36. In the bottom alignment, the second
amino acid sequence (germline Homsap IGKV4-1*01 F) is SEQ ID NO:37.
In the bottom alignment, the third sequence is the CDR3 of a P95C1
light chain variable region and is SEQ ID NO:15.
DETAILED DESCRIPTION
A. Definitions
Unless otherwise defined, all technical terms used herein have the
same meaning as commonly understood by one of ordinary skill in the
art to which this invention belongs.
An anti-LAM antibody may take one of numerous forms in the art, as
disclosed herein. Antibodies are in part defined by the antigens to
which they bind, thus, an "anti-LAM antibody" is any such antibody
which specifically binds at least one epitope of lipoarabinomannan
(LAM) as described herein. It is understood in the art that an
antibody is a glycoprotein comprising at least two heavy (H) chains
and two light (L) chains inter-connected by disulfide bonds, or an
antigen binding portion thereof. A heavy chain is comprised of a
heavy chain variable region (VH) and a heavy chain constant region
(CH1, CH2 and CH3). A light chain is comprised of a light chain
variable region (VL) and a light chain constant region (CL). The
variable regions of both the heavy and light chains comprise
framework regions (FWR) and complementarity determining regions
(CDR). The four FWR regions are relatively conserved while CDR
regions (CDR1, CDR2 and CDR3) represent hypervariable regions and
are arranged from NH2 terminus to the COOH terminus as follows:
FWR1, CDR1, FWR2, CDR2, FWR3, CDR3, FWR4. The variable regions of
the heavy and light chains contain a binding domain that interacts
with an antigen while, depending of the isotype, the constant
region(s) may mediate the binding of the immunoglobulin to host
tissues or factors.
An anti-PIM6/LAM antibody may take one of numerous forms in the
art, as disclosed herein. An "anti-PIM6/LAM antibody" is any such
antibody which specifically binds at least one epitope that is
shared by phosphatidylinositol mannoside 6 (PIM6) and LAM as
described herein. A human mAb specific for an epitope shared by LAM
and PIM6 described herein is P95C1 which binds specifically to at
least one polymannose structure in PIM6 and in the PIM6 related
mannan domain of LM and LAM. P95C1 binds to both LAM and PIM6
because it sees a common (shared) epitope, and is thus referred to
herein as an "anti-PIM6/LAM antibody" or "anti-PIM6/LAM monoclonal
antibody," "human anti-PIM6/LAM antibody" or "human anti-PIM6/LAM
monoclonal antibody."
It is known in the art that it is possible to manipulate monoclonal
and other antibodies and use techniques of recombinant DNA
technology to produce other antibodies or chimeric molecules which
retain the specificity of the original antibody. Such techniques
may evolve introducing DNA encoding the immunoglobulin variable
region, or CDRs, of an antibody to the constant regions, or
constant regions plus framework regions, of a different
immunoglobulin.
The term "antibody" (Ab) as used herein is used in the broadest
sense and specifically may include any immunoglobulin, whether
natural or partly or wholly synthetically produced, including but
not limited to monoclonal antibodies, polyclonal antibodies,
multispecific antibodies (for example, bispecific antibodies and
polyreactive antibodies), and antibody fragments. Thus, the term
"antibody" as used in any context within this specification is
meant to include, but not be limited to, any specific binding
member, immunoglobulin class and/or isotype (e.g., IgG1, IgG2a,
IgG2b, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE) and biologically
relevant fragment or specific binding member thereof, including but
not limited to Fab, F(ab').sub.2, scFv (single chain or related
entity) and (scFv).sub.2.
The term "antibody fragments" as used herein may include those
antibody fragments obtained using techniques readily known and
available to those of ordinary skill in the art, as reviewed
herein. Therefore, the term "antibody" describes any polypeptide or
protein comprising a portion of an intact antibody, such as the
antigen binding or variable region of the intact antibody. These
can be derived from natural sources, or they may be partly or
wholly synthetically produced. Examples of antibody fragments
include, but are not limited to, Fab, Fab', F(ab')2, and Fv
fragments; diabodies, and linear antibodies. In particular, as used
herein, "single-chain Fv" ("sFv" or "scFv") are antibody fragments
that comprise the VH and VL antibody domains connected into a
single polypeptide chain. The sFv polypeptide can further comprise,
e.g., a linker such as a flexible polypeptide linker between the VH
and VL domains that enables the scFv to form the desired structure
for antigen binding.
The term "monoclonal antibody" or "mAb" as used herein may refer to
an antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that may be present in minor amounts.
The terms "variants," "derivatives," and/or "variants and/or
derivatives" as used herein may refer to antibodies, antibody
fragments, recombinant antibodies, whether derived from natural
sources or partly or wholly synthetically produced, as well as
proteins, protein fragments, and polypeptides, inasmuch as the
foregoing compounds are either structurally similar, i.e. retain a
degree of identity that is at least 50%, at least 55%, at least
60%, at least 65%, at least 70%, at least 80%, at least 85%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least
99%, or greater sequence identity with an original unmodified
antibody, and/or, independent of structural identity, may be
functionally similar to the original unmodified anti-LAM and
anti-PIM6/LAM antibodies, that is, they retain the ability to
specifically bind to at least one epitope of LAM or to the shared
PIM6/LAM epitope, respectively. For example, such variants and/or
derivatives may include anti-LAM or anti-PIM6/LAM antibodies with
variant Fc domains, chimeric antibodies, fusion proteins,
bispecific antibodies, or other recombinant antibodies. Such
variants and/or derivative antibodies may, but not necessarily,
possess greater binding specificity for one or more epitope(s) of
LAM, or PIM6, and/or may be able to bind to additional LAM or PIM6
epitopes.
The term "biological sample" refers to a sample obtained from an
organism (e.g., patient) or from components (e.g., cells) of an
organism. The sample may be of any biological tissue, cell(s) or
fluid. The sample may be a "clinical sample" which is a sample
derived from a subject, such as a human patient. Such samples
include, but are not limited to, saliva, sputum, blood, blood cells
(e.g., white cells), amniotic fluid, plasma, semen, bone marrow,
and tissue or fine needle biopsy samples, urine, peritoneal fluid,
and pleural fluid, or cells therefrom. Biological samples may also
include sections of tissues such as frozen sections taken for
histological purposes. A biological sample may also be referred to
as a "patient sample." A biological sample may also include a
substantially purified or isolated protein, membrane preparation,
or cell culture.
The terms "effective amount" or "therapeutically effective amount"
as used herein may refer to an amount of the compound or agent that
is capable of producing a medically desirable result in a treated
subject. The treatment method can be performed in vivo or ex vivo,
alone or in conjunction with other drugs or therapy. A
therapeutically effective amount can be administered in one or more
administrations, applications or dosages and is not intended to be
limited to a particular formulation or administration route.
The term "antigen binding fragment" or "Fab" as used herein may
refer to a region on an antibody that binds to antigens. One of
ordinary skill in the art will understand that Fabs are comprised
of one constant and one variable domain of each of the heavy and
light chain of an antibody.
As used herein, the terms "specific binding," "selective binding,"
"selectively binds," and "specifically binds," may refer to
antibody binding to an epitope on a predetermined antigen but not
to other antigens. Typically, the antibody (i) binds with an
equilibrium dissociation constant (K.sub.D) of approximately less
than 10.sup.-6 M, such as approximately less than 10.sup.-7 M,
10.sup.-8 M, 10.sup.-9 M or 10.sup.-10 M or even lower when
determined by, e.g., surface plasmon resonance (SPR) technology in
a BIACORE.RTM. 2000 surface plasmon resonance instrument using the
predetermined antigen, e.g., a LAM epitope, as the analyte and the
antibody as the ligand, or Scatchard analysis of binding of the
antibody to antigen positive cells, and (ii) binds to the
predetermined antigen with an affinity that is at least two-fold
greater than its affinity for binding to a non-specific antigen
(e.g., BSA, casein) other than the predetermined antigen or a
closely-related antigen.
The terms "conservative sequence modifications" or "conservative
substitutions" as used herein may refer to amino acid modifications
that do not significantly affect or alter the binding
characteristics of the antibody containing the amino acid sequence.
Such conservative modifications include amino acid substitutions,
additions and deletions. Modifications can be introduced into an
antibody of the invention by standard techniques known in the art,
such as site-directed mutagenesis and PCR-mediated mutagenesis.
Conservative amino acid substitutions are ones in which the amino
acid residue is replaced with an amino acid residue having a
similar side chain. Families of amino acid residues having similar
side chains have been defined in the art. These families include
amino acids with basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteine, tryptophan),
nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,
proline, phenylalanine, methionine), beta-branched side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one
or more amino acid residues within the CDR regions of an antibody
of the invention can be replaced with other amino acid residues
from the same side chain family and the altered antibody can be
tested for retained function using the functional assays described
herein.
The term "identity" as used herein may refer to the existence of
shared structure between two compositions. The term "identity" in
the context of proteins may refer to the amount (e.g. expressed in
a percentage) of overlap between two or more amino acid and/or
peptide sequences. In the context of nucleic acids, the term may
refer to the amount (e.g. expressed in a percentage) of overlap
between two or more nucleic acid sequences. As used herein, the
percent (%) identity between two sequences is equivalent to the
percent identity between the two sequences. The percent identity
between the two sequences is a function of the number of identical
positions shared by the sequences (i.e., % identity=# of identical
positions/total # of positions.times.100), taking into account the
number of gaps, and the length of each gap, which need to be
introduced for optimal alignment of the two sequences. The
comparison of sequences and determination of percent identity
between two sequences can be accomplished using a mathematical
algorithm. Such identity is well-represented in the art via local
alignment tools and/or algorithms, and may include pairwise
alignment, multiple sequence alignment methods, structural
alignment methods, and/or phylogenetic analysis methods. Specific
examples include the following. The percent identity between two
amino acid sequences can be determined using the algorithm of E.
Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) which
has been incorporated into the ALIGN program (version 2.0), using a
PAM120 weight residue table, a gap length penalty of 12 and a gap
penalty of 4. In addition, the percent identity between two amino
acid sequences can be determined using the Needleman and Wunsch (J.
Mol. Biol. 48:444-453 (1970)) algorithm which has been incorporated
into the GAP program in the GCG software package (available at
www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix,
and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight
of 1, 2, 3, 4, 5, or 6. Additionally or alternatively, the protein
sequences of the present invention can further be used as a "query
sequence" to perform a search against public databases to, for
example, identify related sequences. Such searches can be performed
using the XBLAST program (version 2.0) of Altschul, et al. (1990)
J. Mol. Biol. 215:403-10. BLAST protein searches can be performed
with the XBLAST program, score=50, wordlength=3 to obtain amino
acid sequences homologous to the antibody molecules of the
invention. To obtain gapped alignments for comparison purposes,
Gapped BLAST can be utilized as described in Altschul et al.,
(1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST
and Gapped BLAST programs, the default parameters of the respective
programs (e.g., XBLAST and NBLAST) can be used.
The terms "co-administration," "co-administered," and "in
combination with" as used herein may refer to the administration of
at least two agents or therapies to a subject. In some embodiments,
the co-administration of two or more agents/therapies is
concurrent. In other embodiments, a first agent/therapy is
administered prior to a second agent/therapy. Those of skill in the
art understand that the formulations and/or routes of
administration of the various agents/therapies used may vary.
The term "carriers" as used herein may include pharmaceutically
acceptable carriers, excipients, or stabilizers that are nontoxic
to the cell or mammal being exposed thereto at the dosages and
concentrations employed. Often the physiologically acceptable
carrier is an aqueous pH buffered solution. Examples of
physiologically acceptable carriers include, but not limited to,
buffers such as phosphate, citrate, and other organic acids;
antioxidants including, but not limited to, ascorbic acid; low
molecular weight (less than about 10 residues) polypeptide;
proteins, such as, but not limited to, serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as, but not limited to,
polyvinylpyrrolidone; amino acids such as, but not limited to,
glycine, glutamine, asparagine, arginine or lysine;
monosaccharides, disaccharides, and other carbohydrates including,
but not limited to, glucose, mannose, or dextrins; chelating agents
such as, but not limited to, EDTA; sugar alcohols such as, but not
limited to, mannitol or sorbitol; salt-forming counterions such as,
but not limited to, sodium; and/or nonionic surfactants such as,
but not limited to, TWEEN; polyethylene glycol (PEG), and
PLURONICS.
The term "treating" or "treatment" of a disease refers to executing
a protocol, which may include administering one or more drugs to a
patient (human or otherwise), in an effort to alleviate signs or
symptoms of the disease. Alleviation can occur prior to signs or
symptoms of the disease appearing as well as after their
appearance. Thus, "treating" or "treatment" includes "preventing"
or "prevention" of disease. The terms "prevent" or "preventing"
refer to prophylactic and/or preventative measures, wherein the
object is to prevent or slow down the targeted pathologic condition
or disorder. For example, in the case of infection by a virulent
strain of the Mycobacterium tuberculosis-complex, "preventing" or
"preventing" may arise in a situation where a course of treatment
is advanced in order to prevent or stall infection by a virulent
strain of the Mycobacterium tuberculosis-complex, such as through
vaccination or passive administration of a protective antibody.
Such "preventing" or "prevention" also arise in the case of latent
infection by Mycobacterium tuberculosis, in which the object would
be to prevent active infection and/or clear a patient of said
latent infection. In addition, "treating" or "treatment" does not
require complete alleviation of signs or symptoms, does not require
a cure, and specifically includes protocols that have only a
marginal effect on the patient.
The terms "patient," "subject" and "individual" are used
interchangeably herein and may refer to a biological system to
which a treatment can be administered. A biological system can
include, for example, an individual cell, a set of cells (e.g., a
cell culture), an organ, a tissue, or a multi-cellular organism. A
"patient," "subject" or "individual" can refer to a human patient,
subject or individual or a non-human patient, subject or
individual.
The term "epitope" as used herein may refer to the region of an
antigen to which an antibody or T cell binds. An "antigen" refers
to a substance that elicits an immunological reaction or binds to
the products of that reaction.
As used herein, the term "vector" means a nucleic acid molecule
capable of transporting another nucleic acid to which it has been
linked. Vectors capable of directing the expression of genes to
which they are operatively linked are referred to herein as
"expression vectors."
As used herein, "protein" and "polypeptide" are used synonymously
to mean any peptide-linked chain of amino acids, regardless of
length or post-translational modification, e.g., glycosylation or
phosphorylation.
The term "labeled," with regard to an antibody, nucleic acid,
peptide, polypeptide, cell, or probe, is intended to encompass
direct labeling of the antibody, nucleic acid, peptide,
polypeptide, cell, or probe by coupling (i.e., physically linking)
a detectable substance to the antibody, nucleic acid, peptide,
polypeptide, cell, or probe.
The terms "purified" or "isolated" peptide, polypeptide, or protein
refers to a peptide, polypeptide, or protein, as used herein, may
refer to a peptide, polypeptide, or protein that has been separated
from other proteins, lipids, and nucleic acids with which it is
naturally associated. The polypeptide/protein can constitute at
least 10% (i.e., any percentage between 10% and 100%, e.g., 20%,
30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, and 99%) by dry weight
of the purified preparation. Purity can be measured by any
appropriate standard method, for example, by column chromatography,
polyacrylamide gel electrophoresis, or HPLC analysis. An isolated
polypeptide/protein (e.g., anti-LAM antibodies) described in the
invention can be produced by recombinant DNA techniques.
B. Mycobacterium tuberculosis
Tuberculosis (TB) remains one of the world's deadliest communicable
diseases, currently infecting approximately one-third of the
world's population. An estimated 9.0 million people developed TB in
2013, and an estimated 1.5 million people died from the disease.
Although there currently are antibiotic treatments available, these
require lengthy treatments, and are increasingly compromised by the
development of multi-drug resistant (MDR-TB) strains, which
currently are responsible for about 3.5% of recent infections.
These strains are much harder to treat and have significantly
poorer cure rates. Also spreading are extensively drug-resistant TB
(XDR-TB) strains, which are even more expensive and difficult to
treat than MDR-TB strains, and have now been reported in 100
countries around the world.
There is a long-established paradigm that immunity against TB
relies solely on cellular defense mechanisms. However, studies in
the HIV field highlight the remarkable ability of the human humoral
immune system to generate diverse antibodies with remarkable
neutralization breadth and potency, and the present invention
highlights the ability of the humoral immune system to produce high
affinity antibodies that recognize multiple LAM epitopes. This
suggests that much of the past difficulty in demonstrating an
important role for antibody-mediated protection against TB may be
due to the limitations in the quality and source of the antibodies
used in past studies, and that applying methods of the present
invention to generate more highly evolved antibodies from
chronically-infected human patients may illustrate the critical
role of the humoral response in immunity to TB.
Some embodiments of the invention are directed to methods for the
in vitro culture of memory B cells from infected humans and
molecular cloning of the variable regions of IgG heavy (H) and
light (L) chains from a single cell. These methods may be utilized
to generate human monoclonal antibodies against the major surface
antigen LAM. The present invention relates to such antibodies, and
engineered derivatives of these antibodies, that possess unique
epitope specificities and binding properties, and the
immunodiagnostic and immunotherapeutic applications of these
antibodies.
C. Lipoarabinomannan (LAM)
One prominent antigenic target of the antibodies of the present
invention is the surface glycolipid, lipoarabinomannan (LAM), a
major structural component of the cell wall of Mycobacterium
tuberculosis-complex members. The present invention identifies a
previously unappreciated heterogeneity in the antigenic structure
of LAM and in the humoral immune response towards LAM in response
to infection and immunization. The structure of LAM is detailed in
Khoo et al., "Variation in Mannose-capped Terminal Arabinan Motifs
of Lipoarabinomannan from Clinical Isolates of Mycobacterium
tuberculosis and Mycobacterium avium Complex," Journal of
Biological Chemistry Vol 276, No. 6, Feb. 9, 2001, incorporated by
reference herein in its entirety. The structure of LAM is complex,
exhibiting an overall tripartite structure with four distinct
structural domains; a phosphatidylinositol lipid anchor
(Mannonsyl-Phosphatidyl-myo-Inositol), an
.alpha.(1.fwdarw.6)-linked D-mannan backbone with terminal
.alpha.(1.fwdarw.2)-Manp-linked side chains, an D-arabinan chain
containing multiple tetra-/hexa-arabinofuranoside branches, and
various capping motifs. LAM consists of a heterogeneous population
of molecules, which can be resolved into multiple isoforms that
possess different biological properties. This heterogeneity is due
to varying lengths of the mannan and arabino chains, different
branching patterns, different numbers of such branches, and
modification of the arabino-side chains by mannose capping, MTX
addition and acylation by fatty acids, succinates and lactates.
Virulent strains of the Mycobacterium tuberculosis-complex are
extensively capped with mono-, di-, and
tri-.alpha.(1.fwdarw.2)-D-Manp saccharide units, while fast growing
non-pathogenic strains like M. smegmatis have uncapped ends or
phosphatidyl-myo-inositol caps (PILAM). It has been estimated that
40-70% of the nonreducing termini of LAM from pathogenic strains of
the Mycobacterium tuberculosis-complex are mannose-capped, and
analysis of the relative abundance of the different cap motifs for
the virulent MT103 clinical strain showed that the dimannosyl unit
was the major structural motif (75-80%), while the mannosyl and the
trimannosyl motifs were present at lower concentrations (10-13%).
This extensive capping may present a unique marker to differentiate
virulent strains of the Mycobacterium tuberculosis-complex from
non-virulent/non-pathogenic strains, such as M. smegmatis, and may
also provide potential antigenic targets for therapeutic use of the
anti-LAM antibodies of the present invention. In addition, some of
the terminal mannose sugars in ManLAM found in the strain M.
tuberculosis are further modified by .alpha.(1.quadrature.4)
addition of a unique structure, 5-deoxy-5-methyl-thio-pentofuranose
(MTX), which affects the immunoreactivity towards different mAbs
sensitive to capping motifs, such as A194-01 and P30B9; MTX
addition increases reactivity with A194-01 and decreases reactivity
towards P30B9. This substitution is present in low abundance, and
may present a unique marker to identify M. tuberculosis,
potentially even from other virulent members of the Mycobacterium
tuberculosis-complex, such as from M. bovis and M. africanum, and
may also provide a potential antigenic target for therapeutic use
of the anti-LAM antibodies of the present invention.
Secreted forms of LAM are important targets for immunodiagnostic
assays of infection by pathogenic members of the M.
tuberculosis-complex. In addition, a considerable body of evidence
indicates that LAM is an important mediator of a number of
functions that promote productive infection and pathogenicity. LAM
is involved in maintaining cell wall integrity and resistance to
beta-lactam antibiotics. Reduced expression of LAM on the bacterial
surface correlated with defective macrophage entry, inhibition of
phagosome-lysosome fusion, attenuation in macrophages, and
increased sensitivity to adaptive immunity, and the binding of
terminal mannosyl units of ManLAM to the mannose receptor on the
surface of macrophages has been described as a critical step in the
uptake of mycobacteria into phagocytic cells. Without wishing to be
bound by theory, it is believed that ManLAM interacts with the
C-type lectins, such as dendritic cell-specific intercellular
adhesion molecule-3 (ICAM-3) grabbing non-integrin (DC-SIGN) the
macrophage mannose receptor (MMR) and Dectin-2 on dendritic cells.
Once inside macrophages, LAM is believed to inhibit
phagosome-lysosome fusion which would lead to the destruction of
the bacteria, thereby allowing the bacteria to persist inside the
macrophages.
LAM is also secreted from the surface of bacteria, and the
extracellular LAM binds to dendritic cell-surface receptors,
including DC-SIGN and Dectin-2. These interactions are believed to
suppress dendritic cell function and interfere with the host immune
system, contributing to immune evasion. Because LAM is in
relatively large quantities during active infection, it can be
detected in the blood and urine of infected patients, for example,
by one or more anti-LAM antibodies of the present invention. These
may be used, for example, in diagnostic kits and methods related to
said diagnostic kits.
D. Anti-LAM and Anti-PIM6/LAM Antibodies
The anti-LAM antibodies of the present invention may comprise
isolated, cultured, or engineered variants and/or derivatives of
human monoclonal antibodies that recognize at least one epitope on
lipoarabinomannan (LAM). An anti-PIM6/LAM antibody (e.g., P95C1) as
described herein specifically binds at least one polymannose
structure in PIM6 and in the PIM6 cross-reactive mannan domain of
LAM. The anti-LAM and anti-PIM6/LAM antibodies of the present
invention may be purified according to methods known in the art.
Such methods may include, for example but not limited to, affinity
chromatography, ion exchange chromatography, immobilized metal
chelate chromatography, thiophilic adsorption, physiochemical
fractionation, or other antigen-specific affinity methods, for
example, methods including protein A, G, and L antibody-binding
ligands. Such purified antibodies may or may not have structural
characteristics that are different from human monoclonal antibodies
that are not purified. For example, conformational epitope changes
for human monoclonal antibodies may occur upon purification.
Antibodies may be bound to additional molecules that are removed
upon purification. Accordingly, such purified antibodies may or may
not have different functional activity. The anti-LAM and
anti-PIM6/LAM antibodies of the present invention may have a number
of structural modifications. For example, the anti-LAM and
anti-PIM6/LAM antibodies of the present invention may be
glycosylated, PEGylated, or otherwise chemically modified in such a
manner as to affect the stability, function, bioavailability,
epitope recognition, or other functional activity. The anti-LAM and
anti-PIM6/LAM antibodies of the present invention may be engineered
variants and/or derivatives of those antibodies described below,
and may or may not possess functional or structural equivalence.
Accordingly, such variants and/or derivatives are still considered
within the scope of the present invention, so long as they are
derived or engineered at least in part from an isolated human
monoclonal anti-LAM or anti-PIM6/LAM antibody, and/or recognize at
least one epitope on LAM.
1. A194-01
In some embodiments, the present invention is directed to the human
monoclonal antibody A194-01 including variants and/or derivatives
thereof. A194-01 is specific for LAM. A194-01 possesses very high
binding activity for LAM, for example, the IgG isotype of A194-01
may exhibit 50% maximal binding activity of the antibody at a
concentration of approximately 20 ng/ml, thus signifying a high
affinity for LAM. A194-01 was originally isolated and purified as
an IgG, however, A194-01 may exist in a number of isotypes, as well
as engineered and recombinant isotypes, including but not limited
to IgG, IgA, IgM, monovalent single chain Fv (scFv) fragments, Fab
proteins, divalent scFv fragments, single chain scFv fragments
(monomers) wherein individual variable light and variable heavy
regions are joined by e.g. a flexible linker, and dimeric scFv
proteins in which two scFv monomers are joined to one another (FIG.
1A) Some particular engineered variants and/or derivatives of
A194-01 include, but are not limited to the following. One
engineered variant and/or derivative of A194-01 comprises a
tetravalent scFv-IgG, formed by joining the A194-01 scFv antigen to
the N-terminus of A194-01 IgG (FIG. 1B, FIG. 17), which may
increase binding affinity and broaden the range of epitopes
recognized (examples of this are given in FIGS. 14 and 15). The
tetravalent scFv-IgG may comprise leader-VH-VL-IgG, or may comprise
leader-VL-VH-IgG. One having ordinary skill in the art will
appreciate that engineered scFv-IgG variants and/or derivatives may
have valences beyond just tetravalent. Another engineered variant
and/or derivative of A194-01 comprises a pentavalent IgM, generated
by converting a dimeric A194-01 IgG to a human IgM contain domain,
wherein such pentavalent IgM contains 10 binding sites (FIG. 1B).
One of ordinary skill in the art will appreciate that further
combinations of A194-01 antigenic fragments are possible and are
considered within the scope of this invention, particularly those
antibody fragments that display complementarity determining regions
(CDRs) specific to A194-01.
The IgG isotype of A194-01 recognizes a unique and complex epitope
that is expressed on unmodified Ara4 and Ara6 side-chains and on
side-chains bearing a single mannose. Although A194-01 does not
recognize side chains bearing di- or tri-mannose substitutions, it
does react with such structures if they are further modified with
an MSX substituent. Accordingly, the IgG isotype of A194-01 binds
to PILAM and ManLAM with high affinity, and also binds strongly
with uncapped versions of both Ara4 and Ara6 structures, and binds
somewhat less strongly to single mannose-capped and MSX-substituted
Ara4/Ara6 structures, but poorly if at all to di-substituted and
tri-substituted ManLAM (FIG. 4). Without wishing to be bound by
theory, the dramatically different effect of attachment of mannose
versus MSX to the terminal mannose of the mono-mannosylated Ara4
structure may reflect a difference between the .alpha.(1.fwdarw.2)
linkage of the mannose and .alpha.(1.fwdarw.4) linkage of the MSX
substitution. Engineered variants and/or derivatives of A194-01,
including those that possess higher valencies, may exhibit broader
epitope specificity than the A194-01 IgG isotype (FIG. 14), and may
further exhibit enhanced affinity for LAM (FIG. 15). For example,
the tetravalent scFv-IgG engineered A194-01 and the engineered IgA
and IgM isotypes bind to both Ara4 and Ara6 structures with higher
affinity than the A194-01 IgG isotype, and furthermore, also
recognize di-mannose and tri-mannose capped structures that the IgG
isotype binds to poorly (FIG. 14). Because pathogenic species of
the Mycobacterium tuberculosis-complex predominantly exhibit
di-mannose capped structures, these engineered variants and/or
derivatives of A194-01, including scFv-IgG and IgM isotypes, may
prove particularly useful for diagnostic kits and methods, as well
as for therapeutic use.
Further engineered variants and/or derivatives of A194-01 include
those antibodies wherein the IgG1 Fc domain is converted to IgG3,
which is more opsogenic, or by generating multimeric versions, by
substituting the IgG1 constant domain by dimeric IgA or pentameric
or hexameric IgM. Without wishing to be bound by theory, this may
significantly enhance avidity of the anti-LAM antibodies by
increasing the flexibility and range of bivalent and multivalent
binding, which contributes to affinity (FIG. 1). This is of
potential clinical significance, as treatments would be
particularly valuable in cases of exposure or infection with MDR or
X-MDR strains of Myocbacterium tuberculosis, which cannot be
effectively treated with traditional antibiotics.
TABLE-US-00001 TABLE 1 A194-01 Complementarity Determining Regions
(CDR) Light Chain CDR1- RSIRSA (SEQ ID NO: 1) CDR2- GAS (SEQ ID NO:
2) CDR3- QQYDFWYTF (SEQ ID NO: 3) Heavy Chain CDR1- GFNFEDFG (SEQ
ID NO: 4) CDR2- ISWNGANI (SEQ ID NO: 5) CDR3- IDWYRDDYYKMDV (SEQ ID
NO: 6)
One of ordinary skill in the art will appreciate that CDRs are
crucial to the diversity of antigen specificities. One having
ordinary skill in the art will further appreciate that CDR3 is the
most variable of CDR regions, and as such bears the greatest
importance, with diversity in the CDR3 region of the variable heavy
chain being sufficient for most antibody specificities.
Accordingly, in some embodiments, the anti-LAM antibodies have a
CDR1, CDR2, and CDR3 region of the variable light chain as set
forth in SEQ ID NOS: 1, 2 and 3, respectively. In some embodiments,
the anti-LAM antibodies have a CDR1, CDR2, and CDR3 region of the
variable light chain as set forth in SEQ ID NOS: 1, 2, and 3
respectively with conservative sequence modifications. In some
embodiments, the anti-LAM antibodies have a CDR1, CDR2, and CDR3
region of the variable light chain having up to 95% identity with
SEQ ID NOS: 1, 2, and 3 respectively. In other embodiments, the
anti-LAM antibodies have a CDR1, CDR2, and CDR3 region of the
variable light chain having up to 90% identity with SEQ ID NOS: 1,
2, and 3 respectively. In other embodiments, the anti-LAM
antibodies have a CDR1, CDR2, and CDR3 region of the variable light
chain having up to 85% identity with SEQ ID NOS: 1, 2, and 3
respectively. In other embodiments, the anti-LAM antibodies have a
CDR1, CDR2, and CDR3 region of the variable light chain having up
to 80% identity with SEQ ID NOS: 1, 2, and 3 respectively. In some
embodiments, the anti-LAM antibodies have a CDR3 region of the
variable light chain as set forth in SEQ ID NO: 3. In some
embodiments, the anti-LAM antibodies have a CDR3 region of the
variable light chain as set forth in SEQ ID NO: 3 with conservative
sequence modifications. In other embodiments, the anti-LAM
antibodies have a CDR3 region of the variable light chain having up
to 95% identity with SEQ ID NO: 3. In other embodiments, the
anti-LAM antibodies have a CDR3 region of the variable light chain
having up to 90% identity with SEQ ID NO: 3. In other embodiments,
the anti-LAM antibodies have a CDR3 region of the variable light
chain having up to 85% identity with SEQ ID NO: 3. In other
embodiments, the anti-LAM antibodies have a CDR3 region of the
variable light chain having up to 80% identity with SEQ ID NO:
3.
In some embodiments, the anti-LAM antibodies have a CDR1, CDR2, and
CDR3 region of the variable heavy chain as set forth in SEQ ID NOS:
4, 5 and 6, respectively. In some embodiments, the anti-LAM
antibodies have a CDR1, CDR2, and CDR3 region of the variable heavy
chain as set forth in SEQ ID NOS: 4, 5 and 6, respectively with
conservative sequence modifications. In some embodiments, the
anti-LAM antibodies have a CDR1, CDR2, and CDR3 region of the
variable heavy chain having up to 95% identity with SEQ ID NOS: 4,
5, and 6 respectively. In other embodiments, the anti-LAM
antibodies have a CDR1, CDR2, and CDR3 region of the variable heavy
chain having up to 90% identity with SEQ ID NOS: 4, 5, and 6
respectively. In other embodiments, the anti-LAM antibodies have a
CDR1, CDR2, and CDR3 region of the variable heavy chain having up
to 85% identity with SEQ ID NOS: 4, 5, and 6 respectively. In other
embodiments, the anti-LAM antibodies have a CDR1, CDR2, and CDR3
region of the variable heavy chain having up to 80% identity with
SEQ ID NOS: 4, 5, and 6 respectively. In some embodiments, the
anti-LAM antibodies have a CDR3 region of the variable heavy chain
as set forth in SEQ ID NO: 6. In some embodiments, the anti-LAM
antibodies have a CDR3 region of the variable heavy chain as set
forth in SEQ ID NO: 6 with conservative sequence modifications. In
other embodiments, the anti-LAM antibodies have a CDR3 region of
the variable heavy chain having up to 95% identity with SEQ ID NO:
6. In other embodiments, the anti-LAM antibodies have a CDR3 region
of the variable heavy chain having up to 90% identity with SEQ ID
NO: 6. In other embodiments, the anti-LAM antibodies have a CDR3
region of the variable heavy chain having up to 85% identity with
SEQ ID NO: 6. In other embodiments, the anti-LAM antibodies have a
CDR3 region of the variable heavy chain having up to 80% identity
with SEQ ID NO: 6.
In the experiments described herein, the A194-01 antibody was
expressed by transfection of H and L chain vectors in Expi293 cells
and cultured in standard Expi293 serum-free medium for several
days. The secreted antibody was purified from the culture
supernatant by affinity chromatography on columns conjugated with
Protein A or Protein G ligands. The bound antibodies were released
from the ligands by treatment with low pH buffer (0.2 M
glycine-HCl, pH 2.5) and neutralized with 1/50 volume of 2 M tris
buffer buffer (pH 8.8). The buffer was exchanged with PBS by
dialysis or by several rounds of concentration on centrifugal
filters (Amicon Ultra centrifugal filters, 30K mw limit).
The amino acid (aa) and nucleic acid (nt) sequences for A194 heavy
and light chain sequences are as follows:
TABLE-US-00002 A194 Heavy chain nt sequence: (SEQ ID NO: 39)
CAAGTGCAGCTGTTGGAGTCTGGGGGAGGTGTGGTACGGCCGGGGGGGTC
CCTGAGACTCTCCTGTGCAGCCTCTGGATTCAACTTTGAAGATTTTGGCA
TGAGCTGGGTCCGCCAAGCTCCAGGGAAGGGGCTGGAGTGGGTCTCTAGT
ATTAGTTGGAATGGTGCTAATATAGGCTATGTAGACTCTGTGAAGGGCCG
ATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTATATCTGCAAATGA
ACAGTCTGAGAGCCGAGGACACGGCCTTATATTACTGTGCGATAGACTGG
TACAGAGACGACTACTACAAGATGGACGTCTGGGGCAAAGGGACCACGGT
CACCGTCTCCTCAGCCTCGACCAAGGGCCCATCGGTCTTCCCGCTAGCGC
CCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTC
AAGGACTACTTCCCCGAACCTGTGACGGTCTCGTGGAACTCAGGCGCCCT
GACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCT
ACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAG
ACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAA
GAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCC
CAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAA
CCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGT
GGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGG
ACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTAC
AACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTG
GCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAG
CCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCA
CAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGT
CAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGG
AGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCC
GTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGA
CAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATG
AGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGT AAATGA A194
Heavy chain aa sequence: (SEQ ID NO: 40)
QVQLLESGGGVVRPGGSLRLSCAASGFNFEDFGMSWVRQAPGKGLEWVSS
ISWNGANIGYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAIDW
YRDDYYKMDVWGKGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ
TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK
PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY
NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP
QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K* A194 Light
chain nt sequence (kappa): (SEQ ID NO: 41)
GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTCTCTCCAGGGGA
AAGAGCCACCCTCTCCTGCAGGGCCAGTCGGAGTATTCGCAGCGCCTTAG
CCTGGTACCAGCACAAACCTGGCCAGGCTCCCAGGCTCCTCATCTTTGGT
GCATCCACCAGGGCCACTGGTATCCCAGCCAGGTTCAGTGGCAGTGGGTC
TGGGACAGACTTCACTCTCACCGTCAGCAGCATACGGTCTGAGGATTCTG
CAGTTTATTACTGTCAGCAGTATGATTTCTGGTACACTTTTGGCCAGGGG
ACCAAGCTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTT
CCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCC
TGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTCGAC
AACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAG
CAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAG
ACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTG
AGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG A194 Light chain aa
sequence (kappa): (SEQ ID NO: 42)
EIVMTQSPATLSVSPGERATLSCRASRSIRSALAWYQHKPGQAPRLLIFG
ASTRATGIPARFSGSGSGTDFTLTVSSIRSEDSAVYYCQQYDFWYTFGQG
TKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD
NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL
SSPVTKSFNRGEC*
2. P30B9
In some embodiments, the present invention is directed to the
recombinant human monoclonal antibody P30B9 including variants
and/or derivatives thereof. P30B9 is specific for LAM. P30B9 was
originally isolated and purified as an IgM, however, P30B9 may
exist in a number of isotypes, as well as engineered and
recombinant isotypes, including but not limited to IgM, IgG, IgA,
as well as antigenic fragments thereof, including but not limited
to monovalent single chain Fv (scFv) fragments, Fab proteins,
divalent scFv fragments, single chain scFv fragments (monomers)
wherein individual variable light and variable heavy regions are
joined by e.g. a flexible linker, and dimeric scFv proteins in
which two scFv monomers are joined to one another.
The IgM isotype of P30B9 binds most potently to di-mannose
substituted Ara4 and Arab LAM epitopes with the
Manp-.alpha.(1.fwdarw.2)-Manp-(1.fwdarw.5)-Araf structures (FIGS.
4, 16 and 18) although other Manp-.alpha. substituted structures
(e.g., structure 2, 4 and 59 in FIG. 8) may also be recognized with
lower affinities. The preferential recognition of P30B9 for
di-mannose capped LAM has potential clinical relevance, since
di-mannose caps are reported to be the dominant LAM modification on
virulent strains of the Mycobacterium tuberculosis-complex. Without
wishing to be bound by theory, it is believed that terminal
mannosyl units mediate binding of LAM from virulent strains of the
Mycobacterium tuberculosis-complex to human macrophage and other
immune cells that leads to the perturbation of immune function and
establishment of stable infection. Without wishing to be bound by
theory, binding of the mannose caps to the mannose receptor is
believed to limit phagosome-lysosome (P-L) fusion and facilitate
survival of the bacterium in infected macrophages. The specificity
of P30B9 for di-mannose capped LAM is indicated by the specificity
of this mAb for glyconjugates bearing this structure, and in the
fact that the IgM isotype of P30B9 binds specifically to LAM
derived from either Mycobacterium tuberculosis, but not to LAM from
Mycobacterium smegmatis or Mycobacterium leprae, which do not
contain di-mannose capped LAM epitopes. This is in contrast to the
IgG isotype of A194-01, which binds to PILAM, uncapped Ara4/Ara6
residues, and mono-mannose capped LAM epitopes, all of which are
common in Mycobacterium smegmatis and Mycobacterium leprae. Like
the IgM isotype of P30B9, the IgM isotype of A194-01 is able to
bind to di-mannose and tri-mannose capped LAM epitopes (FIG. 14),
presumable due to an increased binding avidity.
Therefore, the IgM isotypes of P30B9 may serve as an important
immunodiagnostic reagent for detecting infection by virulent
members of the Mycobacterium tuberculosis-complex and
distinguishing said virulent members other nonpathogenic
mycobacterial species, as it is specific to di-mannose capped LAM.
Furthermore, the IgM isotype of the P30B9 antibody as well as
engineered variants and/or derivatives of A194-01 may possess
immunotherapeutic activity that limit infection and pathogenesis of
virulent members of the Mycobacterium tuberculosis-complex and may
be suitable for use in therapy, either in combination with
traditional antibiotics, additional antibodies, or alone, or may be
used as a passive immunotherapeutic agent. The IgM isotype of P30B9
binds specifically to ManLAM derived from Mycobacterium
tuberculosis with high affinity (FIG. 2A,B), but not to PILAM
derived from Mycobacterium smegmatis (FIG. 2C).
Engineered variants and/or derivatives of P30B9 may include, for
example, P30B9 expressed in the IgA isotype, including dimeric IgA1
and IgA2. Without wishing to be bound by theory, it is believed
that polyvalency is required for P30B9 function, as this antibody
was isolated as an IgM, and is not active when expressed as an IgG.
The present invention shows that P30B9 is active in engineered IgA
isotypes, including dimeric IgA1 and IgA2. This was tested by
moving the P30B9 VH domain into IgA1 and IgA2 vectors. IgA1 differs
from IgA2 mostly by the presence of a 16 amino-acid insertion,
comprised of a repeat of 8 amino acids rich in proline, serine, and
threonine, and modified with 3-6, O-linked oligosaccharides [FIG.
5]. The binding activity of the engineered IgA forms of P30B9 to
ManLAM were compared to those of the IgG and IgM forms. The IgM
form had the highest activity, while both of the IgA forms were
also able to bind to ManLAM, with the IgA2 form showing weaker
activity than the IgA1 form, and the IgG form was inactive in an
ELISA against ManLAM (FIG. 6).
TABLE-US-00003 TABLE 2 P30B9 Complementarity Determining Regions
(CDR) Light Chain CDR1- QSINSN (SEQ ID NO: 7) CDR2- KAS (SEQ ID NO:
8) CDR3- QQYKAFKTF (SEQ ID NO: 9) Heavy Chain CDR1- GGSFSGYY (SEQ
ID NO: 10) CDR2- FDLGGSITHSRGT (SEQ ID NO: 11) CDR3- RGLAMGGTKEFDS
(SEQ ID NO: 12)
One of ordinary skill in the art will appreciate that CDRs are
crucial to the diversity of antigen specificities. One having
ordinary skill in the art will further appreciate that CDR3 is the
most variable of CDR regions, and as such bears the greatest
importance, with diversity in the CDR3 region of the variable heavy
chain being sufficient for most antibody specificities.
Accordingly, in some embodiments, the anti-LAM antibodies have a
CDR1, CDR2, and CDR3 region of the variable light chain as set
forth in SEQ ID NOS: 7, 8 and 9, respectively. In some embodiments,
the anti-LAM antibodies have a CDR1, CDR2, and CDR3 region of the
variable light chain as set forth in SEQ ID NOS: 7, 8 and 9,
respectively with conservative sequence modifications. In some
embodiments, the anti-LAM antibodies have a CDR1, CDR2, and CDR3
region of the variable light chain having up to 95% identity with
SEQ ID NOS: 7, 8, and 9 respectively. In other embodiments, the
anti-LAM antibodies have a CDR1, CDR2, and CDR3 region of the
variable light chain having up to 90% identity with SEQ ID NOS: 7,
8, and 9 respectively. In other embodiments, the anti-LAM
antibodies have a CDR1, CDR2, and CDR3 region of the variable light
chain having up to 85% identity with SEQ ID NOS: 7, 8, and 9
respectively. In other embodiments, the anti-LAM antibodies have a
CDR1, CDR2, and CDR3 region of the variable light chain having up
to 80% identity with SEQ ID NOS: 7, 8, and 9 respectively. In some
embodiments, the anti-LAM antibodies have a CDR3 region of the
variable light chain as set forth in SEQ ID NO: 9. In some
embodiments, the anti-LAM antibodies have a CDR3 region of the
variable light chain as set forth in SEQ ID NO: 9 with conservative
sequence modifications. In other embodiments, the anti-LAM
antibodies have a CDR3 region of the variable light chain having up
to 95% identity with SEQ ID NO: 9. In other embodiments, the
anti-LAM antibodies have a CDR3 region of the variable light chain
having up to 90% identity with SEQ ID NO: 9. In other embodiments,
the anti-LAM antibodies have a CDR3 region of the variable light
chain having up to 85% identity with SEQ ID NO: 9. In other
embodiments, the anti-LAM antibodies have a CDR3 region of the
variable light chain having up to 80% identity with SEQ ID NO:
9.
In some embodiments, the anti-LAM antibodies have a CDR1, CDR2, and
CDR3 region of the variable heavy chain as set forth in SEQ ID NOS:
10, 11 and 12, respectively. In some embodiments, the anti-LAM
antibodies have a CDR1, CDR2, and CDR3 region of the variable heavy
chain as set forth in SEQ ID NOS: 10, 11 and 12, respectively with
conservative sequence modifications. In some embodiments, the
anti-LAM antibodies have a CDR1, CDR2, and CDR3 region of the
variable heavy chain having up to 95% identity with SEQ ID NOS: 10,
11 and 12, respectively. In other embodiments, the anti-LAM
antibodies have a CDR1, CDR2, and CDR3 region of the variable heavy
chain having up to 90% identity with SEQ ID NOS: 10, 11 and 12,
respectively. In other embodiments, the anti-LAM antibodies have a
CDR1, CDR2, and CDR3 region of the variable heavy chain having up
to 85% identity with SEQ ID NOS: 10, 11 and 12, respectively. In
other embodiments, the anti-LAM antibodies have a CDR1, CDR2, and
CDR3 region of the variable heavy chain having up to 80% identity
with SEQ ID NOS: 10, 11 and 12, respectively. In some embodiments,
the anti-LAM antibodies have a CDR3 region of the variable heavy
chain as set forth in SEQ ID NO: 12. In some embodiments, the
anti-LAM antibodies have a CDR3 region of the variable heavy chain
as set forth in SEQ ID NO: 12 with conservative sequence
modifications. In other embodiments, the anti-LAM antibodies have a
CDR3 region of the variable heavy chain having up to 95% identity
with SEQ ID NO: 12. In other embodiments, the anti-LAM antibodies
have a CDR3 region of the variable heavy chain having up to 90%
identity with SEQ ID NO: 12. In other embodiments, the anti-LAM
antibodies have a CDR3 region of the variable heavy chain having up
to 85% identity with SEQ ID NO: 12. In other embodiments, the
anti-LAM antibodies have a CDR3 region of the variable heavy chain
having up to 80% identity with SEQ ID NO: 12.
In the experiments described herein, the P30B9 antibody was
expressed by transfection of H and L chain vectors in Expi293 cells
and cultured in standard Expi293 serum-free medium for several
days. The secreted antibody was purified from the culture
supernatant by affinity chromatography on columns conjugated with
Protein L ligand. The bound antibody was released from the ligands
by treatment with low pH buffer (0.2 M glycine-HCl, pH 2.5) and
neutralized with 1/50 volume of 2 M tris buffer buffer (pH 8.8).
The buffer was exchanged with PBS by dialysis or by several rounds
of concentration on centrifugal filters (Amicon Ultra centrifugal
filters, 30K mw limit).
The amino acid sequences for P30B9 heavy chain and light chain and
their comparison with its closest germline are shown in FIG. 22.
The amino acid and nucleotide sequences for P30B9 including the
CDR3 region are copied below:
TABLE-US-00004 P30B9-Heavy chain variable region: (SEQ ID NO: 43)
QVQLQQWGAGLLKPSETLSLTCAVY GGSFSGYY WSWIRQSPETGLEW LGE FDLGGS
ITHSRGT NYNPSLKSRVTISGDTSKNQFSLKLTSVTAA DTAVYYC ARGLAMGGTKEFDS
P30B9-Light chain variable region: (SEQ ID NO: 44)
DIQMTQSPDSLSASVGDRITITCRAS QSINSN LAWYQQKPGKAPKLLI Y KAS
DLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QQYKAFK T P30B9-Heavy chain
DNA sequence: (SEQ ID NO: 45)
caggtgcagctacagcagtggggcgcaggactgttgaagccttcggagac
cctgtccctcacctgcgctgtctatggtgggtccttcagtggttactact
ggagctggatccgccagtccccagagacggggctggagtggcttggcgaa
TTCGATCTTGGTGGAAGCatcactcatagtagaggcaccaactacaaccc
gtcgctcaagagtcgagtcaccatctcaggagacacgtccaagaaccagt
tctccctgaaactgacctctgtgaccgccgcggacacggctgtctattac
tgtgcgagaggtttagcaatgggtggaactaaggagtttgactcctgggg
ccagggaaccctggtcaccgtctcctcag P30B9-Light chain: (SEQ ID NO: 46)
gacatccagatgacccagtctccagactccctgtctgcatctgtaggaga
cagaatcaccatcacttgccgggccagtcagagtattaatagtaatttgg
cctggtatcagcagaaaccggggaaagcccctaagctcctgatctataag
gcgtctgatttagaaagtggggtcccatcaaggttcagcggcagtggatc
tgggacagaattcactctcaccatcagcagcctgcagcctgatgattttg
caacttattattgccaacagtataaagcattcaagacgttcggccacggg
accaaggtggaaatcaaac
3. P95C1
In some embodiments, the present invention is directed to the
recombinant human monoclonal antibody P95C1 including variants
and/or derivatives thereof. P95C1 is specific for an epitope shared
by LAM, LM and PIM6. Although P95C1 was originally isolated and
purified as an IgM, P95C1 is also active when expressed in other
isotypes, including but not limited to IgG and IgA forms.
One of ordinary skill in the art will appreciate that CDRs are
crucial to the diversity of antigen specificities. One having
ordinary skill in the art will further appreciate that CDR3 is the
most variable of CDR regions, and as such bears the greatest
importance, with diversity in the CDR3 region of the variable heavy
chain being sufficient for most antibody specificities.
Accordingly, in some embodiments, the anti-LAM antibodies have a
CDR1, CDR2, and CDR3 region of the variable light chain as set
forth in SEQ ID NOS: 13, 14 and 15, respectively. In some
embodiments, the anti-LAM antibodies have a CDR1, CDR2, and CDR3
region of the variable light chain as set forth in SEQ ID NOS: 13,
14 and 15, respectively with conservative sequence modifications.
In some embodiments, the anti-LAM antibodies have a CDR1, CDR2, and
CDR3 region of the variable light chain having up to 95% identity
with SEQ ID NOS: 13, 14 and 15, respectively. In other embodiments,
the anti-LAM antibodies have a CDR1, CDR2, and CDR3 region of the
variable light chain having up to 90% identity with SEQ ID NOS: 13,
14 and 15, respectively. In other embodiments, the anti-LAM
antibodies have a CDR1, CDR2, and CDR3 region of the variable light
chain having up to 85% identity with SEQ ID NOS: 13, 14 and 15,
respectively. In other embodiments, the anti-LAM antibodies have a
CDR1, CDR2, and CDR3 region of the variable light chain having up
to 80% identity with SEQ ID NOS: 13, 14 and 15, respectively. In
some embodiments, the anti-LAM antibodies have a CDR3 region of the
variable light chain as set forth in SEQ ID NO: 15. In some
embodiments, the anti-LAM antibodies have a CDR3 region of the
variable light chain as set forth in SEQ ID NO: 15 with
conservative sequence modifications. In other embodiments, the
anti-LAM antibodies have a CDR3 region of the variable light chain
having up to 95% identity with SEQ ID NO: 15. In other embodiments,
the anti-LAM antibodies have a CDR3 region of the variable light
chain having up to 90% identity with SEQ ID NO: 15. In other
embodiments, the anti-LAM antibodies have a CDR3 region of the
variable light chain having up to 85% identity with SEQ ID NO: 15.
In other embodiments, the anti-LAM antibodies have a CDR3 region of
the variable light chain having up to 80% identity with SEQ ID NO:
15.
In some embodiments, the anti-LAM antibodies have a CDR1, CDR2, and
CDR3 region of the variable heavy chain as set forth in SEQ ID NOS:
16, 17 and 18, respectively. In some embodiments, the anti-LAM
antibodies have a CDR1, CDR2, and CDR3 region of the variable heavy
chain as set forth in SEQ ID NOS: 16, 17 and 18, respectively with
conservative sequence modifications. In some embodiments, the
anti-LAM antibodies have a CDR1, CDR2, and CDR3 region of the
variable heavy chain having up to 95% identity with SEQ ID NOS: 16,
17 and 18, respectively. In other embodiments, the anti-LAM
antibodies have a CDR1, CDR2, and CDR3 region of the variable heavy
chain having up to 90% identity with SEQ ID NOS: 16, 17 and 18,
respectively. In other embodiments, the anti-LAM antibodies have a
CDR1, CDR2, and CDR3 region of the variable heavy chain having up
to 85% identity with SEQ ID NOS: 16, 17 and 18, respectively. In
other embodiments, the anti-LAM antibodies have a CDR1, CDR2, and
CDR3 region of the variable heavy chain having up to 80% identity
with SEQ ID NOS: 16, 17 and 18, respectively. In some embodiments,
the anti-LAM antibodies have a CDR3 region of the variable heavy
chain as set forth in SEQ ID NO: 18. In some embodiments, the
anti-LAM antibodies have a CDR3 region of the variable heavy chain
as set forth in SEQ ID NO: 18 with conservative sequence
modifications. In other embodiments, the anti-LAM antibodies have a
CDR3 region of the variable heavy chain having up to 95% identity
with SEQ ID NO: 18. In other embodiments, the anti-LAM antibodies
have a CDR3 region of the variable heavy chain having up to 90%
identity with SEQ ID NO: 18. In other embodiments, the anti-LAM
antibodies have a CDR3 region of the variable heavy chain having up
to 85% identity with SEQ ID NO: 18. In other embodiments, the
anti-LAM antibodies have a CDR3 region of the variable heavy chain
having up to 80% identity with SEQ ID NO: 18.
TABLE-US-00005 TABLE 3 P95C1 Complimentary Determining Regions
(CDR) Light Chain CDR1: QNVLDSANNRNY (SEQ ID NO: 13) CDR2: WAS (SEQ
ID NO: 14) CDR3: TQYHRLPHT (SEQ ID NO: 15) Heavy Chain CDR1:
GGSINTNNW (SEQ ID NO: 16) CDR2: IHRHGDT (SEQ ID NO: 17) CDR3:
CPLGYCSGDDCHRVA (SEQ ID NO: 18)
The P95C1 IgM/.kappa. antibody was originally identified in
supernatants of BCL6/Bcl-xL transduced memory B cells and cloned
from these cells into IgM/.kappa. expression vectors using the
standard RT-PCR protocol. The antibody was expressed by
transfection of H and L chain vectors in Expi293 cells and cultured
in standard Expi293 serum-free medium for several days. The
secreted antibody was purified from the culture supernatant by
affinity chromatography on columns conjugated with Protein L
ligand. The bound antibody was released from the ligands by
treatment with low pH buffer (0.2 M glycine-HCl, pH 2.5) and
neutralized with 1/50 volume of 2 M tris buffer buffer (pH 8.8).
The buffer was exchanged with PBS by dialysis or by several rounds
of concentration on centrifugal filters (Amicon Ultra centrifugal
filters, 30K mw limit).
The amino acid sequences for P95C1 heavy chain and light chain and
their comparison with its closest germline are shown in FIG. 23.
The amino acid and nucleotide sequences for P95C1 including the
CDR3 region are copied below:
TABLE-US-00006 P95C1-Heavy chain variable region: (SEQ ID NO: 47)
EVQLLESGPGLVRPWGTLSLTCAVS GGSINTNNW WSWVRQSPGKGLEW IGE IHRHGDT
NYNPSLKRRVSISMDESMNQFSLRLISVTAADTAVYYC CPLGYCSGDDCHRVA P95C1-Light
chain variable region: (SEQ ID NO: 48) DIQMTQSPSSLSVSLGERATINCKSS
QNVLDSANNRNY FGWYQQKPGQ PPKLLIS WAS
TRESGVPDRFSGSGSGTDFTLIISGLQVEDVAVYYC TQYHRLPHT P95C1-Heavy chain:
(SEQ ID NO: 49) gaggtgcagctcttggagtcgggcccaggactggtgaggccttgggggac
tctgtccctcacctgcgctgtctctggtggctccatcaatactaataact
ggtggagttgggtccgccagtccccggggaaggggctggagtggattgga
gaaatccatcgtcatggggacaccaactacaacccgtcactcaagaggcg
agtctccatatcgatggacgagtccatgaaccagttctccctgaggctta
tctctgtgaccgccgcggacacggccgtgtattactgttgtcccctagga
tattgtagtggtgatgactgtcaccgagttgcctggggccggggaatcct
ggtcaccgtctcttcag P95C1-Light chain: (SEQ ID NO: 50)
gacatccagatgacccagtctccatcctccctgtctgtgtctctgggcga
gagggccaccatcaactgcaagtccagccagaatgttttagacagcgcca
acaataggaactacttcggttggtaccagcagaaaccagggcagcctcct
aagctgctcatttcctgggcatctacacgggaatccggggtccctgaccg
attcagtggcagcggctctgggacagacttcactctcatcatcagcggcc
tgcaggttgaagatgtggcagtttattactgtacacagtatcatagactt
cctcacaccttcggccaagggacacgactggaaattaaac
E. Further Variants and/or Derivatives
One of ordinary skill in the art will appreciate that given the CDR
regions of A194-01, P30B9, and P95C1, a wide number of engineered
variants and/or derivatives of the anti-LAM antibodies disclosed
herein may be constructed. For example, the anti-LAM antibodies of
the present invention may be engineered into chimeric antibodies,
humanized antibodies, and chimeric/humanized antibodies that
exhibit affinity to one or more LAM epitopes. The antibodies may be
engineered into bispecific antibodies, or may be engineered such
that a single antibody construct binds to multiple LAM
epitopes.
As described herein, the anti-LAM antibodies of the present
invention may be engineered as homologous scFv-IgG constructs or as
heterologous scFv-IgG constructs. Homolgous scFv-IgG constructs of
A194-01 are detailed in this Application (FIG. 17A). One
non-limiting example of a heterologous scFv-IgG construct would be
where the VH and VL chains of P30B9 were joined to the A194-01 IgG
by a linker (FIG. 17B), although other VH/VL chains could be used,
for example other anti-LAM antibodies such as murine anti-LAM
antibodies. This may allow recognition of distinct epitopes in a
single antigen molecule and may enhance multivalent binding and
lead to increased affinity. Alternatively, heterologous scFv-IgG
constructs may generate bispecific antibodies if the additional
VH/VL chains target an antigen other than LAM.
The anti-LAM antibodies of the present invention may also be
engineered to create scFv-IgM constructs, including both homologous
and heterologous scFv-IgM constructs. A non-limiting example of a
homologous scFv-IgM would be where P30B9 VH/VL chains are joined to
the P30B9 IgM. In this construct, all binding sites would possess
the same epitope specificity. A non-limiting example of a
heterologous scFv-IgM construct is where the A194-01 scFv is joined
to the P30B9 IgM, as opposed to the IgG constant domain
[non-limiting [FIG. 17C]. Such engineered variant and/or derivative
construct would retain the IgM-dependent recognition of dimannose
epitopes of the parental P30B9 mAb and add the additional binding
specificity of the A194-01 scFv. This may allow recognition of
unique epitope arrays and lead to enhanced affinities, which could
be valuable for improved point-of-care antigen detection
assays.
F. Diagnostic Kits and Methods
One embodiment of the present invention relates to diagnostic kits
and methods for the detection and/or quantification of LAM and/or
PIM6 in a sample. As described herein, the anti-LAM antibodies
A194-01 and P30B9, as well as the anti-PIM6/LAM antibody P95C1,
including engineered variants and/or derivatives thereof, may be
effective in detecting and/or quantifying the amount of LAM and/or
PIM6 present in a sample. The LAM or PIM6 may be derived from any
source, such as from Mycobacterium tuberculosis or Mycobacterium
smegmatis, or from a serum or urine sample from a patient, e.g. a
patient infected with a virulent strain of the Mycobacterium
tuberculosis-complex. The LAM may be e.g. PILAM, ManLAM, or
uncapped/unmodified AraLAM from other mycobacterial strains, such
as M. leprae. These strains differ in the nature and extent of
capping that occurs, and different antibody combinations would
therefore have different specificities for the different forms,
allowing some level of differentiation or typing to be performed.
In particular, the IgM and engineered IgA1 isotype of P30B9, as
well as the engineered IgM and scFv-IgG isotypes of A194-01, would
be well-suited for detecting and/or quantifying di-mannose
substituted ManLAM in a sample from a TB patient, which in some
circumstances may comprise 80% of said LAM, and various isotypes of
P30B9 would be especially effective at detecting and/or quantifying
LAM bearing di-mannose substituted Ara6 residues, which as
described herein are particularly prevalent on LAM derived from
Mycobacterium tuberculosis. Since the P95C1 epitope is highly
conserved in all species of LAM, this antibody, when coupled with a
second antibody with the proper specificity, would be well-suited
for detecting and/or quantifying various types of LAM in a sample.
The IgG isotype of A194-01 binds very effectively to various forms
of LAM, especially unsubstituted LAM, mono-mannosylated LAM, and
PILAM, and so would be effective at detecting and/or quantifying
LAM derived from various strains of mycobacteria. The engineered
IgM and scFv-IgG isotypes would also be quite effective at
detecting and/or quantifying the amount of unsubstituted LAM,
mono-mannosylated LAM, and PILAM, and additionally may bind to di-
and tri-mannose substituted LAM. This endows the engineered
variants and/or derivatives of A194-01 with greater epitope
recognition than the IgG isotype of A194-01 or the IgM isotype of
P30B9, but at the expense of specificity for only those LAM
epitopes that are specific to virulent strains of Mycobacterium
tuberculosis. In some embodiments, quantifying said specificity for
LAM and/or PIM6 is achieved by comparing the signal intensity of a
serially diluted control sample having a known concentration of LAM
and/or PIM6 in various direct binding assays or or antigen-capture
assays.
Because the IgG isotype of A194-01, the IgM/IgA isotypes of P30B9,
and the various isotypes of P95C1 bind to different LAM epitopes
that are variably expressed in different strains of Mycobacterium
tuberculosis, these particular isotypes could be used to
differentiate the origination of a source of LAM; di-mannose
substituted LAM, in particular di-mannose substituted Arab residues
comprise the majority of LAM residues in virulent strains of
Mycobacterium tuberculosis, whereas unsubstituted LAM/PILAM
residues comprise the majority of LAM residues in fast growing
non-virulent strains such as Mycobacterium smegmatis. For example,
samples comprising LAM that bind only to A194-01 IgG and not P30B9
IgM likely did not originate from a virulent strain of
Mycobacterium tuberculosis, whereas samples that bind to both P30B0
IgM and A194-01 IgG likely did originate from a virulent strain of
Mycobacterium tuberculosis or a species of mycobacteria that
introduces a similar capping motif.
Because the IgM/IgA isotypes of P30B9 are specific for di-mannose
substituted ManLAM, which as detailed herein is the dominant form
in virulent strains of Mycobacterium tuberculosis, said isotypes of
P30B9 are ideal candidates for diagnostic kits and methods of use
for diagnosing a patient as being infected with a virulent strain
of the Mycobacterium tuberculosis-complex. Furthermore, the
engineered IgM and scFv-IgG variants and/or derivatives of A194-01
may be suitable for such a use as they also recognize di-mannose
and tri-mannose substituted ManLAM epitopes. Such a patient could
have an ongoing or active infection, or the infection could be
latent. The strain could be multi-drug resistant (MDR) or could be
extensively-drug resistant (XDR). Specifically regarding patients
having latent infections, change in LAM concentration in the serum
or urine may be of particular importance, as increases in
concentrations may signify a change to active infection.
Alternatively, a decrease in concentration in an individual who has
an active infection may signify that treatment is effective and
should be continued, or an increase in concentration during
treatment may indicate that the current treatment is not effective
and should be eliminated, changed and/or modified.
The methods for diagnosing infection may including contacting a
biological sample from said patient, e.g. blood, plasma, urine,
sputum, or other bodily fluid, with at least one anti-LAM antibody
and/or at least one anti-PIM6/LAM antibody of the present
invention, particularly those anti-LAM antibodies that recognize
di-mannose substituted ManLAM and those anti-PIM6/LAM antibodies
that recognize at least one polymannose structure in the PIM6
mannan domain. These include, for example, IgM and IgA isotypes of
P30B9, the engineered IgA, IgM and scFv-IgG isotypes of A194-01,
and the various isotypes (IgG, IgM, IgA) of P95C1.
The antibodies used as the detecting reagent may be bound to
reporter molecules such as those known in the art. The antibodies
may be part of a kit, e.g. bound to a substrate or part of a
sandwich assay. The kits may include a first anti-LAM or
anti-PIM6/LAM capture antibody, a second anti-LAM or anti-PIM6/LAM
detector (detection) antibody which is bound to a reporter
molecule, and a support to which the capture anti-LAM or
anti-PIM6/LAM antibody is bound. The first and second anti-LAM
antibody may bind to the same LAM epitopes which are present in
multiple copies on a single LAM molecule, or preferably they may
bind to different epitopes present on a single LAM molecule. The
LAM and PIM6 epitopes may be any of those described herein. The
kits may include a third capture or detector (detection) antibody
which binds to a non-competing site of the first and second
antibody. This may increase the number of molecules captured and
number of detector molecules bound and the strength of the
corresponding signal.
The kits may include instructions for use, and may further contain
various reagents, solvents, diluents, and/or pharmaceutically
acceptable preservatives. The sensitivity of different
biotin-labeled anti-LAM monoclonal antibodies in such an assay was
conducted [FIG. 7]. In this assay, the murine anti-LAM antibody
CS-35 was used to capture ManLAM from solution. This antibody was
selected because of its broad specificity. CS-35 (250 ng/well) was
used to capture ManLAM from solutions containing differing
concentrations, and different biotinylated monoclonal antibodies
were then used to probe for the presence of ManLAM in the capture
well. Using a cut-off of 3.times.SD of background, the most
sensitive probe was A194-01 IgM, which gave a strong signal (1.8
OD) for the highest dilution of ManLAM (0.016 ng/well). This was
superior to the two FIND murine antibodies, which have been
previously considered to be the best available probes for this type
of assay.
G. Therapeutic Compositions, Methods, Vaccines, and Vectors
One embodiment of the present invention is directed towards
pharmaceutical compositions comprising at least one anti-LAM
antibody or anti-PIM6/LAM antibody of the present invention, as
well as their methods of use in treating a patient in need thereof.
The patient may have a latent or active infection by a virulent
strain of Mycobacterium tuberculosis, and of particular utility,
the strain may be multi-drug resistant (MDR) or extensively drug
resistant (XDR) to traditional therapies/antibiotics. The anti-LAM
and anti-PIM6/LAM antibodies utilized in these compositions and
methods may be any anti-LAM antibody or anti-PIM6/LAM antibody of
the present invention, but of particular utility may be those
anti-LAM antibodies that recognize di-mannose capped ManLAM,
particularly di-mannose capped Ara6 residues, e.g. P30B9 IgM or
IgA1/IgA2 isotype and pentavalent A194-01 IgM or tetravalent
scFv-IgG isotype and various isotypes of P95C1.
A pharmaceutically acceptable anti-LAM antibody and/or
anti-PIM6/LAM antibody composition suitable for patient
administration will contain an effective amount of the anti-LAM or
anti-PIM6/LAM antibody or antibodies in a formulation which both
retains biological activity while also promoting maximal stability
during storage within an acceptable temperature range. The
pharmaceutical compositions can also include, depending on the
formulation desired, pharmaceutically acceptable diluents,
pharmaceutically acceptable carriers and/or pharmaceutically
acceptable excipients, or any such vehicle commonly used to
formulate pharmaceutical compositions for animal or human
administration. The diluent is selected so as not to affect the
biological activity of the combination. Examples of such diluents
are distilled water, physiological phosphate-buffered saline,
Ringer's solutions, dextrose solution, and Hank's solution. The
amount of an excipient that is useful in the pharmaceutical
composition or formulation of this invention is an amount that
serves to uniformly distribute the antibody throughout the
composition so that it can be uniformly dispersed when it is to be
delivered to a subject in need thereof. It may serve to dilute the
antibody to a concentration which provides the desired beneficial
palliative or curative results while at the same time minimizing
any adverse side effects that might occur from too high a
concentration. It may also have a preservative effect. Thus, for
the antibody having a high physiological activity, more of the
excipient will be employed. On the other hand, for any active
ingredient(s) that exhibit a lower physiological activity, a lesser
quantity of the excipient will be employed.
The pharmaceutically acceptable anti-LAM antibody and/or
anti-PIM6/LAM antibody composition may be in liquid form or solid
form. A solid formulation is generally lyophilized and brought into
solution prior to administration for either single or multiple
dosing. The formulations should not be exposed to extreme
temperature or pH so as to avoid thermal denaturation. Thus, it is
essential to formulate an antibody composition of the present
invention within a biologically relevant pH range. A solution
buffered to maintain a proper pH range during storage is indicated,
especially for liquid formulations stored for longer periods of
time between formulation and administration. To date, both liquid
and solid formulations require storage at lower temperatures
(usually 2-8.degree. C.) in order to retain stability for longer
periods. Formulated antibody compositions, especially liquid
formulations, may contain a bacteriostat to prevent or minimize
proteolysis during storage, including but not limited to effective
concentrations (usually <1% w/v) of benzyl alcohol, phenol,
m-cresol, chlorobutanol, methylparaben, and/or propylparaben. A
bacteriostat may be contraindicated for some patients. Therefore, a
lyophilized formulation may be reconstituted in a solution either
containing or not containing such a component. Additional
components may be added to either a buffered liquid or solid
antibody formulation, including but not limited to sugars as a
cryoprotectant (including but not necessarily limited to
polyhydroxy hydrocarbons such as sorbitol, mannitol, glycerol and
dulcitol and/or disaccharides such as sucrose, lactose, maltose or
trehalose) and, in some instances, a relevant salt (including but
not limited to NaCl, KCl or LiCl). Such antibody formulations,
especially liquid formulations slated for long term storage, will
rely on a useful range of total osmolarity to both promote long
term stability at temperature of 2-8.degree. C., or higher, while
also making the formulation useful for parenteral injection. An
effective range of total osmolarity (the total number of molecules
in solution) is from about 200 mOs/L to about 800 mOs/L. It will be
apparent that the amount of a cyroprotectant, such as sucrose or
sorbitol, will depend upon the amount of salt in the formulation in
order for the total osmolarity of the solution to remain within an
appropriate range. Therefore a salt free formulation may contain
from about 5% to about 25% sucrose, with a preferred range of
sucrose from about 7% to about 15%, with an especially preferred
sucrose concentration in a salt free formulation being from 10% to
12%. Alternatively, a salt free sorbitol-based formulation may
contain sorbitol within a range from about 3% to about 12%, with a
preferred range from about 4% to 7%, and an especially preferred
range is from about 5% to about 6% sorbitol in a salt-free
formulation. Salt-free formulations will of course warrant
increased ranges of the respective cryoprotectant in order to
maintain effective osmolarity levels. These formulation may also
contain a divalent cation (including but not necessarily limited to
MgCl2, CaCl2 and MnCl2); and a non-32 ionic surfactant (including
but not necessarily limited to Polysorbate-80 (Tween 80.RTM.),
Polysorbate-60 (Tween 60.RTM.), Polysorbate-40 (Tween 40.RTM.) and
Polysorbate-20 (Tween 20.RTM.), polyoxyethylene alkyl ethers,
including but not limited to Brij 58.RTM., Brij 35.RTM., as well as
others such as Triton X-100.RTM., Triton X 114.RTM., NP40.RTM.),
Span 85 and the Pluronic series of non-ionic surfactants (e.g.,
Pluronic 121)). Any combination of such components, including
probable inclusion of a bacteriostat, may be useful to fill the
antibody-containing formulations of the present invention. The
antibody composition of the present invention may also be a
"chemical derivative", which describes an antibody that contains
additional chemical moieties which are not normally a part of the
immunogloblulin molecule (e.g., pegylation). Such moieties may
improve the solubility, half-life, absorption, etc. of the base
molecule. Alternatively, the moieties may attenuate undesirable
side effects of the base molecule or decrease the toxicity of the
base molecule.
Specific embodiments include PLGA microspheres, as discussed herein
and as further known in the art, as well as polymer-based
non-degradable vehicles comprising poly (ethylene-co-vinyl acetate;
PEVAc). Additionally, controlled-release and localized delivery of
antibody-based therapeutic products is reviewed in Grainger, et
al., 2004, Expert Opin. Biol. Ther. 4(7): 1029-1044), hereby
incorporated by reference in its entirety. Suitable microcapsules
capable of encapsulating the antibody may also include
hydroxymethylcellulose or gelatin-microcapsules and polymethyl
methacrylate microcapsules prepared by coacervation techniques or
by interfacial polymerization. See PCT publication WO 99/24061
entitled "Method for Producing IGF-1 Sustained-Release
Formulations," wherein a protein is encapsulated in PLGA
microspheres, this reference which is hereby incorporated herein by
reference in its entirety. In addition, microemulsions or colloidal
drug delivery systems such as liposomes and albumin microspheres,
may also be used. Other preferred sustained-release compositions
employ a bioadhesive to retain the antibody at the site of
administration. As noted above, the sustained-release formulation
may comprise a biodegradable polymer into which the antibody is
disposed, which may provide for non-immediate release.
Non-injectable devices may be described herein as an "implant",
"pharmaceutical depot implant", "depot implant", "non-injectable
depot" or some such similar term. Common depot implants may
include, but are not limited to, solid biodegradable and
non-biodegradable polymer devices (such as an extended polymer or
coaxial rod shaped device), as well as numerous pump systems also
known in the art. Injectable devices are split into bolus
injections (release and dissipation of the drug subsequent to
injection), and repository or depot injections, which provide a
storage reservoir at the site of injection, allowing for
sustained-release of the biological agent over time. A depot
implant may be surgically tethered to the point of delivery so as
to provide an adequate reservoir for the prolonged release of the
antibody over time. Such a device will be capable of carrying the
drug formulation in such quantities as therapeutically or
prophylactically required for treatment over the pre-selected
period. The depot implant may also provide protection to the
formulation from degradation by body processes (such as proteases)
for the duration of treatment. As known in the art, the term
"sustained-release" refers to the gradual (continuous or
discontinuous) release of such an agent from the block polymer
matrix over an extended period of time. Regardless of the specific
device, the sustained-release of the anti-LAM antibody and/or
anti-PIM6/LAM antibody composition will result in a local
biologically effective concentrations of the antibody. A sustained
release of the biological agent(s) will be for a period of a single
day, several days, a week or more; but most likely for a month or
more, or up to about six months, depending on the formulation.
Natural or synthetic polymers known in the art will be useful as a
depot implant due to characteristics such as versatile degradation
kinetics, safety, and biocompatibility. These copolymers can be
manipulated to modify the pharmacokinetics of the active
ingredient, shield the agent from enzymatic attack, as well as
degrading over time at the site of attachment or injection. The
artisan will understand that there are ample teachings in the art
to manipulate the properties of these copolymers, including the
respective production process, catalysts used, and final molecular
weight of the sustained-release depot implant or depot injection.
Natural polymers include but are not limited to proteins (e.g.,
collagen, albumin or gelatin); polysaccharides (cellulose, starch,
alginates, chitin, chitosan, cyclodextrin, dextran, hyaluronic
acid) and lipids. Biodegradable synthetic polymers may include but
are not limited to various polyesters, copolymers of L-glutamic
acid and gamma ethyl-L-glutamate (Sidman et al., 1983, Biopolymers
22:547-556), polylactides ([PLA]; U.S. Pat. No. 3,773,919 and EP
058,481), polylactate polyglycolate (PLGA) such as
polylactide-co-glycolide (see, for example, U.S. Pat. Nos.
4,767,628 and 5,654,008), polyglycolide (PG), polyethylene glycol
(PEG) conjugates of poly(.alpha.-hydroxy acids), polyorthoesters,
polyaspirins, polyphosphagenes, vinylpyrrolidone, polyvinyl alcohol
(PVA), PVA-g-PLGA, PEGT-PBT copolymer (polyactive), methacrylates,
poly(N-isopropylacrylamide), PEO-PPO-PEO (pluronics), PEO-PPO-PAA
copolymers, PLGA-PEO-PLGA, polyorthoesters (POE), or any
combinations thereof, as described above (see, for example, U.S.
Pat. No. 6,991,654 and U.S. Pat. Appl. No. 20050187631, each of
which is incorporated herein by reference in its entirety,
hydrogels (see, for example, Langer et al., 1981, J. Biomed. Mater.
Res. 15:167-277; Langer, 1982, Chem. Tech. 12:98-105,
non-degradable ethylene-vinyl acetate (e.g. ethylene vinyl acetate
disks and poly(ethylene-co-vinyl acetate)), degradable lactic
acid-glycolic acid copolymers such as the Lupron Depot.TM.
poly-D-(-)-3-hydroxybutyric acid (EP 133,988), hyaluronic acid gels
(see, for example, U.S. Pat. No. 4,636,524), alginic acid
suspensions, polyorthoesters (POE), and the like. Polylactide (PLA)
and its copolymers with glycolide (PLGA) have been well known in
the art since the commercialization of the Lupron Depot.TM.,
approved in 1989 as the first parenteral sustained-release
formulation utilizing PLA polymers. Additional examples of products
which utilize PLA and PLGA as excipients to achieve
sustained-release of the active ingredient include Amidox (PLA;
periodontal disease), Nutropin Depot (PLGA; with hGH), and the
Trelstar Depot (PLGA; prostate cancer). Other synthetic polymers
included but are not limited to poly(c-caprolactone),
poly3-hydroxybutyrate, poly(.beta.-malic acid) and
poly(dioxanone)]; polyanhydrides, polyurethane (see WO
2005/013936), polyamides, cyclodestrans, polyorthoesters, n-vinyl
alcohol, polyethylene oxide/polyethylene terephthalate,
polyphosphate, polyphosphonate, polyorthoester, polycyanoacrylate,
polyethylenegylcol, polydihydropyran, and polyacytal.
Non-biodegradable devices include but are not limited to various
cellulose derivatives (carboxymethyl cellulose, cellulose acetate,
cellulose acetate propionate, ethyl cellulose, hydroxypropyl methyl
cellulose) silicon-based implants (polydimethylsiloxane), acrylic
polymers, (polymethacrylate, polymethylmethacrylate,
polyhydroxy(ethylmethylacrylate), as well as polyethylene-co-(vinyl
acetate), poloxamer, polyvinylpyrrolidone, poloxamine,
polypropylene, polyamide, polyacetal, polyester, poly
ethylene-chlorotrifluoroethylene, polytetrafluoroethylene (PTFE or
"Teflon.TM."), styrene butadiene rubber, polyethylene,
polypropylene, polyphenylene oxide-polystyrene,
poly-a-chloro-p-xylene, polymethylpentene, polysulfone and other
related biostable polymers. Carriers suitable for sustained-release
depot formulations include, but are not limited to, micospheres,
films, capsules, particles, gels, coatings, matrices, wafers, pills
or other pharmaceutical delivery compositions. Examples of such
sustained-release formulations are described above. See also U.S.
Pat. Nos. 6,953,593; 6,946,146; 6,656,508; 6,541,033; and
6,451,346, the contents of each which are incorporated herein by
reference. The dosage form must be capable of carrying the drug
formulation in such quantities and concentration as therapeutically
required for treatment over the pre-selected period, and must
provide sufficient protection to the formulation from degradation
by body processes for the duration of treatment. For example, the
dosage form can be surrounded by an exterior made of a material
that has properties to protect against degradation from metabolic
processes and the risk of, e.g., leakage, cracking, breakage, or
distortion. This can prevent expelling of the dosage form contents
in an uncontrolled manner under stresses it would be subjected to
during use, e.g., due to physical forces exerted upon the drug
release device as a result of normal joint articulation and other
movements by the subject or for example, in convective drug
delivery devices, physical forces associated with pressure
generated within the reservoir. The drug reservoir or other means
for holding or containing the drug must also be of such material as
to avoid unintended reactions with the active agent formulation,
and is preferably biocompatible (e.g., where the dosage form is
implanted, it is substantially non-reactive with respect to a
subject's body or body fluids). Generally, the respective
biological agent(s) is administered to an individual for at least
12 hours to at least a week, and most likely via an implant
designed to deliver a drug for at least 10, 20, 30, 100 days or at
least 4 months, or at least 6 months or more, as required. The
anti-LAM antibody and/or anti-PIM6/LAM antibody can be delivered at
such relatively low volume rates, e.g., from about 0.001 ml/day to
1 ml/day so as to minimize tissue disturbance or trauma near the
site where the formulation is released. The formulation may be
released at a rate of, depending on the specific biological
agent(s), at a low dose, e.g., from about 0.01 .mu.g/hr or 0.1
.mu.g/hr, 0.25 .mu.g/hr, 1 .mu.g/hr, generally up to about 200
.mu.g/hr, or the formulation is delivered at a low volume rate
e.g., a volume rate of from about 0.001 ml/day to about 1 ml/day,
for example, 0.01 micrograms per day up to about 20 milligrams per
day. Dosage depends on a number of factors such as potency,
bioavailability, and toxicity of the active ingredient (e.g., IgG
antibody) used and the requirements of the subject.
For in vivo treatment of human and non-human patients, the patient
is administered or provided a pharmaceutical formulation including
at least one anti-LAM antibody and/or at least one anti-PIM6/LAM
antibody of the present invention. When used for in vivo therapy,
the anti-LAM or anti-PIM6/LAM antibodies of the invention are
administered to the patient in therapeutically effective amounts
(i.e., amounts that eliminate or reduce the total bacterial load).
The antibodies are administered to a human patient, in accord with
known methods, such as intravenous administration, for example, as
a bolus or by continuous infusion over a period of time, by
intramuscular, intraperitoneal, intracerobrospinal, subcutaneous,
intra-articular, intrasynovial, intrathecal, oral, topical, or
inhalation routes. The antibodies can be administered parenterally,
when possible, at the target cell site, or intravenously. In some
embodiments, antibody is administered by intravenous or
subcutaneous administration. Therapeutic compositions of the
invention may be administered to a patient or subject systemically,
parenterally, or locally. The above parameters for assessing
successful treatment and improvement in the disease are readily
measurable by routine procedures familiar to a physician.
For parenteral administration, the anti-LAM and anti-PIM6/LAM
antibodies may be formulated in a unit dosage injectable form
(solution, suspension, emulsion) in association with a
pharmaceutically acceptable, parenteral vehicle. Examples of such
vehicles include, but are not limited, water, saline, Ringer's
solution, dextrose solution, and 5% human serum albumin.
Non-aqueous vehicles include, but are not limited to, fixed oils
and ethyl oleate. Liposomes can be used as carriers. The vehicle
may contain minor amounts of additives such as substances that
enhance isotonicity and chemical stability, such as, for example,
buffers and preservatives.
The anti-LAM and anti-PIM6/LAM antibodies of the present invention
may be administered to the host in any manner, strategy and/or
combination available in the art in amounts sufficient to offer a
therapeutic treatment against infection by a virulent strain of
Mycobacterium tuberculsosis-complex. These compositions may be
provided to the individual by a variety of routes known in the art,
especially parenteral routes, including but in no way limited to
parenteral routes such as intravenous (IV), intramuscular (IM); or
subcutaneous (SC) administration, with IV administration being the
norm within the art of therapeutic antibody administration. These
compositions may be administered as separate or multiple doses
(i.e., administration of the antibody at staggered times by
maintaining the sterile condition of the formulation through the
treatment regime).
The dose and dosage regimen depends upon a variety of factors
readily determined by a physician, such as the nature of the
infection, for example, its therapeutic index, the patient, and the
patient's history. Generally, a therapeutically effective amount of
an antibody is administered to a patient. In some embodiments, the
amount of antibody administered is in the range of about 0.01 mg/kg
to about 1000 mg/kg of patient body weight, and any range in
between. Depending on the type and severity of the infection, about
0.1 mg/kg to about 50 mg/kg body weight (for example, about 0.1-15
mg/kg/dose) of antibody is an initial candidate dosage for
administration to the patient, whether, for example, by one or more
separate administrations, or by continuous infusion. The progress
of this therapy is readily monitored by conventional methods and
assays and based on criteria known to the physician or other
persons of skill in the art. The above parameters for assessing
successful treatment and improvement in the disease are readily
measurable by routine procedures familiar to a physician.
These antibodies may also be administered via genetic vectors that
express the paired heavy and light chains of a given antibody. This
can involve a plasmid the efficiently expresses these genes or a
viral vector, such as Adenoviral or Adeno-associated virus (AAV)
vectors. These vectors can be delivered by injection into muscle
tissue, and, depending on the dose, can secrete relatively large
amount of secreted antibody into the circulation over a relatively
long period of time.
Other therapeutic regimens may be combined with the administration
of the anti-LAM and/or anti-PIM6/LAM antibodies of the present
invention, for example, with another anti-LAM antibody, including
but not limited to those anti-LAM antibodies known in the art, e.g.
murine anti-LAM antibodies or humanized versions thereof, or with a
pharmaceutical compound, such as, but not limited to, antibiotics.
Antibiotics that are suitable for co-administration with the
anti-LAM and/or anti-PIM6/LAM antibodies of the present invention
include, but are not limited to, isoniazid, rifampin, rifapentine,
ethambutol, pyrazinamide, bedaquiline, capreomycin, cycloserine,
dexamethasone, kanamycin, and tinocordin. The combined
administration includes co-administration, using separate
formulations or a single pharmaceutical formulation, and
consecutive administration in either order, wherein preferably
there is a time period while both (or all) active agents
simultaneously exert their biological activities. Such combined
therapy can result in a synergistic therapeutic effect. The above
parameters for assessing successful treatment and improvement in
the disease are readily measurable by routine procedures familiar
to a physician.
According to another embodiment, the present invention provides a
passive vaccine or pharmaceutical compositions including at least
one anti-LAM and/or anti-PIM6/LAM antibody of the invention and a
pharmaceutically acceptable carrier. According to one embodiment,
the vaccine or pharmaceutical compositions is a composition
including at least one antibody described herein and a
pharmaceutically acceptable carrier. The vaccine can include a
plurality of the antibodies having the characteristics described
herein in any combination and can further include other anti-LAM
antibodies, including those of the present invention and those
known in the art, e.g. murine anti-LAM antibodies or humanized
versions thereof. The passive vaccine may include one or more
pharmaceutically acceptable preservatives, carriers, and/or
excipients, which are known in the art.
According to another embodiment, the present invention covers an
active vaccine or pharmaceutical composition including
administering to patient at least one antigenic LAM or PIM6
epitope. The particular epitope to be employed can be determined by
testing the therapeutic activity of antibodies described in this
patent in an appropriate animal model for TB infection and/or
pathogenesis. This model species can be mouse, or guinea pig, or
rabbit, or primate. For example, ifA194-01 is most protective then
a vaccine bearing a form of the A194-01 epitope would be used,
whereas if P30B9 is most protective, di-mannose substituted Ara6
residues may be most effective at generating an appropriate humoral
response. The active vaccine may include one or more adjuvants,
which are known in the art, e.g. alum, aluminum hydroxide, aluminum
phosphate, paraffin oil, and cytokines, e.g. IL-1, IL-2, IL-12. The
active vaccine may comprise one or more pharmaceutically acceptable
preservatives, carriers, and/or excipients, which are known in the
art.
In some embodiments, the invention is directed to a recombinant
vector, e.g. a plasmid, including a nucleic acid coding for an
immunoglobulin heavy chain (Ig VH) of an anti-LAM antibody or an
anti-PIM6/LAM antibody, and a second nucleic acid coding for an
immunoglobulin light chain (Ig VL). In other embodiments, the first
nucleic acid and the second nucleic acid are in two different
recombinant vectors. According to another embodiment, the present
invention covers a method of treating a tuberculosis infection in
an individual including administering to said individual a first
nucleic acid coding for an immunoglobulin heavy chain (Ig VH) of an
anti-LAM or anti-PIM6/LAM antibody and a second nucleic acid coding
for an immunoglobulin light chain (Ig VL) of an anti-LAM or
anti-PIM6/LAM antibody wherein each of the nucleic acids is
operably linked to a promoter region. The first nucleic acid and
the second nucleic acid may be in a same recombinant vector or in
two different recombinant vectors. The recombinant vector may be
non-replicating viral vectors, e.g. adeno-associated viruses (AAV),
or may be plasmids. In certain embodiments, the invention is
directed to a cell transformed with one or more vectors disclosed
herein.
The above described antibodies and antibody compositions, vaccine
compositions, and vectors can be administered for the prophylactic
and therapeutic treatment of infection by virulent strains of the
Mycobacterium tuberculosis-complex.
H. Equivalents
Where a value of ranges is provided, it is understood that each
intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limit of that range and any other stated or intervening
value in that stated range is encompassed within the invention. The
upper and lower limits of these smaller ranges which may
independently be included in the smaller ranges is also encompassed
within the invention, subject to any specifically excluded limit in
the stated range. Where the stated range includes one or both of
the limits, ranges excluding either both of those included limits
are also included in the invention.
Unless defined otherwise, all technical and scientific terms used
herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can also be used in the practice or testing of the present
invention, the preferred methods and materials are now described.
All publications mentioned herein are incorporated herein by
reference in their entireties.
As used herein and in the appended claims, the singular forms "a",
"and" and "the" include plural references unless the context
clearly dictates otherwise
The term "about" refers to a range of values which would not be
considered by a person of ordinary skill in the art as
substantially different from the baseline values. For example, the
term "about" may refer to a value that is within 20%, 15%, 10%, 9%,
8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the
stated value, as well as values intervening such stated values.
Publications disclosed herein are provided solely for their
disclosure prior to the filing date of the present invention.
Nothing herein is to be construed as an admission that the present
invention is not entitled to antedate such publication by virtue of
prior invention. Further, the dates of publication provided may be
different from the actual publication dates which may need to be
independently confirmed.
Each of the applications and patents cited in this text, as well as
each document or reference, patient or non-patient literature,
cited in each of the applications and patents (including during the
prosecution of each issued patent; "application cited documents"),
and each of the PCT and foreign applications or patents
corresponding to and/or claiming priority from any of these
applications and patents, and each of the documents cited or
referenced in each of the application cited documents, are hereby
expressly incorporated herein by reference in their entirety. More
generally, documents or references are cited in this text, either
in a Reference List before the claims; or in the text itself; and,
each of these documents or references ("herein-cited references"),
as well as each document or reference cited in each of the
herein-cited references (including any manufacturer's
specifications, instructions, etc.), is hereby expressly
incorporated herein by reference.
The following non-limiting examples serve to further illustrate the
present invention.
EXAMPLES
Example 1--the Methods Described Herein were Utilized to Culture
Memory B Cells in vitro and to molecularly clone immunoglobulin
variable region genes to isolate several novel human monoclonal
antibodies (mAbs) specific for LAM. One having ordinary skill in
the art will recognize that these methods described herein can be
adjusted to selectively identify rare antibodies with very high
affinity, which could be present as few as 1 out of 100,000 memory
B cells circulating in the blood of the patient.
Monoclonal Antibodies
Murine monoclonal antibodies: Hybridoma cell lines producing
LAM-specific murine monoclonal antibodies CS-35 and CS-40 obtained
from Dr. Delphi Chatterjee's lab were recloned to homogeneity, and
the antibodies were purified by protein A chromatography.
Antibodies 906.41, 906.7, 908.1 and 922.5 were provided by Dr. John
Spencer, and FIND25 and FIND170 were provided by Tobias Broger at
FIND.
Antigens
Mycobacterium tuberculosis derived H37Rv lipoarabinomannan (LAM)
(NR-14848) and Mycobacterium smegmatis derived LAM (NR-14860) were
obtained from Colorado State University through BEI resources.
LAM-derived glycoconjugates were synthesized in the Lowary lab.
ELISA Assays
Man-LAM (H37Rv) and PI-LAM (derived from Mycobacterium smegmatic)
were diluted in CBC buffer (7.5 mM sodium carbonate, 17.4 mM sodium
bicarbonate, pH 9.0) and plated at a concentration of 100 ng/well
in 96 wells ELISA plates. After overnight incubation of plates at
4.degree. C., wells were washed with PBS, pH 7.4 containing 0.05%
Tween-20 (PBST), then blocked with 1% BSA (Sigma) in PBS buffer.
PBST-washed plates were incubated for 1 hour at 37.degree. C. with
plasma derived from individuals infected with Mycobacterium
tuberculosis and control plasma diluted in RPMI medium containing
2% FBS. PBST washed plates were then incubated for 1 hour with a
1:1000 dilution of alkaline phosphatase conjugated goat-anti-human
IgG (.gamma.-specific) (Millipore), or IgM (.mu.-specific)
(Millipore), or IgA (.alpha.-specific). After PBST washing the
color was developed with 50 .mu.L of DEA buffer. The OD was
measured at 405 nm by spectrophotometer. Titers were defined as the
reciprocal dilution which produced an OD after subtracting the
background OD taken the BSA coated plate, and were determined by
exponential interpolation.
Plasma Titrations
The two purified antigens were obtained from the BEI Repository,
and were plated overnight at 4.degree. C. on 96 well ELISA plates
at a concentration of 2 .mu.g/ml, and the plates were then blocked
with 1% BSA in 1.times.PBS. The LAM-specific titers of plasma were
tested by incubating serially diluted samples at 37.degree. C. for
1 hour, followed which the plates were washed three times with
PBS+0.1% Tween20. Bound antibody was detected with a mixture of
alkaline phosphatase conjugated goat anti-human kappa and goat
anti-human lambda at 1:1,000 dilution in 1% BSA in PBS and the
signals were developed by adding alkaline phosphate substrate in
DEA buffer. Reactivity was measured as OD405 at 30 min.
Human Subjects
Patients with active infection with Mycobacterium tuberculosis were
enrolled in the Lattimore practice at the Global Tuberculosis
Institute. Active infection was defined by culture-proven
tuberculosis disease or a diagnosis of clinical tuberculosis. This
group included patients with a recent tuberculosis diagnosis,
patients who were in the second month of the therapy. Uninfected
patients were HIV-seronegative, tuberculin skin test-negative,
healthy volunteers with no history of Bacillus Calmette-Guerin
(BCG) vaccination and negative for interferon-gamma release assay
(IGRA) (Quantiferon Gold In-Tube, Cellestis Inc, Valencia, Calif.).
Informed written consent was obtained from participants, and the
study was approved by the Rutgers University Institutional Review
Board.
TABLE-US-00007 TABLE 4 Demographics of human subjects Table 4.
Clinical characteristics of TB patients used in this study Sample
ID Bleed date Treatment start date Diagnosis Level of disease TB194
Mar. 3, 2014 Jan. 15, 2014 TST (+), AFB Smear (-), NAAT (+)
Pulmonary TB TB210 Apr. 2, 2014 Mar. 12, 2014 TST (+), AFB Smear
(+), Abnormal X-Ray Pulmonary TB TB256 Jun. 30, 2014 May 19, 2014
TST (+), TBD (+), Abnormal X-Ray Pulmonary TB TB260 Jun. 10, 2014
Jul. 3, 2014 TBD (+), AFB Smear (+), Abnormal X-Ray Pulmonary TB
HC261 Jul. 3, 2014 NA LTBI (-), TST (-) LTBI (-), Non-contact TB310
Nov. 13, 2014 Oct. 11, 2014 TBD (-), AFB Smear (-), IGRA (+)
Pulmonary TB TB314 Nov. 18, 2014 Oct. 13, 2014 TBD (-), AFB Smear
(-), IGRA (+) Pulmonary TB TB320 Dec. 1, 2014 Oct. 31, 2014 TBD
(+), TST (+), Abnormal X-Ray Pulmonary TB TB366 Apr. 10, 2015 Mar.
17, 2015 TBD (+), TST (+), Abnormal X-Ray Pulmonary TB TB372 Apr.
15, 2015 Feb. 27, 2015 TBD (+), TST (+), Abnormal X-Ray Pulmonary
TB TB373 Apr. 22, 2015 Mar. 3, 2015 TBD (+), TST (+), Abnormal
X-Ray Pulmonary TB TB384 May 5, 2015 Apr. 6, 2015 TBD (+), TST (+),
Abnormal X-Ray Pulmonary TB
1. Culture and Isolation of A194-01 (IgG Isotype)
Human monoclonal anti-LAM antibody A194-01, isotype IgG, was
isolated from cultured memory B cells obtained from a TB-infected
patient, TB-194. A critical component of the in vitro culture
system is the presence of suitable feeder cells that can provide
stimulation by CD40L, the ligand for CD40, a member of the
TNF-receptor superfamily that is expressed on the surface of B
cells and plays an essential role in mediating T cell-dependent
immunoglobulin class switching and memory B cell development.
Memory B cells were seeded on a feeder layer of CD40L-expressing
MS40L-low cells. These cells express a low level of CD40L, and have
been previously shown to efficiently support the replication of
memory B cells and their maturation to plasma cells (Luo, X., et
al., Blood, 2009. 113(7). These cells were generated by infecting
murine stromal MS5 cells, that provide the B-lineage growth factor
IL-7, with FUW-CD40L, a virus that transduces human CD40L,
originally obtained from Origene (Rockville, MID). Memory B cells
were isolated with the MACS human memory B cell isolation kit from
Miltenyi (Cat. #130-093-546). Non-B cells were excluded from PBMCs
by negative selection with magnetic beads containing antibodies
against the cell surface marker CD2, CD3, CD14, CD16, CD36, CD43,
CD56, CD66b and glycophorin A. To further eliminate naive B cells,
memory B cell subpopulations were positively selected with magnetic
beads coupled to antibody against the cell surface marker CD27, a
marker for memory B cells that is also expressed in low levels on
plasma cells, but not on naive B cells. In the presence of
CD40L-expressing feeder cells, these conditions support the
replication of the memory B cells and their differentiation into
plasmablasts secreting relatively high titers of Igs into culture
supernatants.
Cultures were refed at weekly intervals by replacing half of the
culture supernatant with fresh media. After 2-3 weeks there were
sufficient B cells to produce .about.1-5 .mu.g/ml of secreted
antibody. Assuming the presence of 100-1,000 distinct clones in
each well, this corresponded to an average concentration of 1-10
ng/mL of Ig per B cell clone. This concentration is fairly low, and
therefore this method was biased towards antibodies with relatively
high affinities for the target antigens. Approximately 80,000 cells
memory B cells were purified from the blood of this patient and
cultured in 96 wells of a 96 well culture plate, for an initial
density of .about.800 cells/well.
Culture supernatants were screened by ELISA for the presence of
antibodies against Mycobacterium tuberculosis-derived LAM. LAM was
coated at a concentration of 2 .mu.g/mL in 50 .mu.L of bicarbonate
coating buffer per well of a 96 wells ELISA plate and incubated at
4.degree. C. overnight. The plate was washed with PBST (0.1% Tween
20 in 1.times.PBS) 4 times, and blocked with 200 .mu.L of 2% nonfat
milk in 1.times.PBS for 1 hour at 37.degree. C. 100 .mu.l of the
culture supernatant was added to the corresponding wells of the
ELISA plate containing LAM, and incubated for 1 hour at 37.degree.
C. After additional washing steps, AP-conjugated mouse anti-human
Fab-antibody was added to detect bound human antibody. After half
an hour of incubation at 37.degree. C., 100 .mu.L of AP-substrate
in DEA buffer was added to the ELISA wells and reactivity was
determined colorimetrically by measuring absorbance at 405 nm.
A positive signal (OD of .about.1 at 1 hr) was detected in only 1
well out of 96 wells, indicating the rarity of these cells in this
sample. Cells from the positive cells were re-cultured at a density
of 5-10 cells/well in 10 wells of 96 well plates and rescreened for
activity against LAM. This resulted in .about.6 positive wells (OD
of .about.1 at 1 hr), again consistent with the low frequency of
LAM-reactive cells and suggesting that the original positive well
contained only a single LAM-positive B cell clone. Cells from
several of the positive sub-clones were lysed and used to isolate
the variable regions of the H and L chains, which were then cloned
into H and L chain expression vectors. A total of 10 diverse VH and
9 VL sequences were isolated from these wells, and these were then
tested for activity by transfecting individual combinations in 293
cells. Of 90 combinations tested, only a single combination of
heavy chain (p9045-IgG1-VH) and light chain (p9044-Vk) gave a
positive signal against LAM. Antibodies were expressed by
cotransfection of corresponding heavy and light chain plasmids in
Expi-292 cells as described by the manufacturer and grown in
serum-free media. Antibodies were purified by affinity
chromatography on either protein A beads (for IgG) or protein L
beads (for IgG), and eluted with low pH buffer. The purified
antibodies were concentrated and characterized by SS-PAGE for size
and purity.
2. Isolation and Culturing of the IgM Isotype of P30B9
Human monoclonal anti-LAM antibody P30B9, isotype IgM, was isolated
from cultured memory B cells obtained from a TB-infected patient,
TB-314. PBMCs were isolated from the blood of patient TB-314 by
centrifugation on a ficoll gradient, and .about.30,000 memory B
cells were purified as described above with the MACS human memory B
cell isolation kit from Miltenyi. The purified memory B cells were
cultured for 14 days by plating at 400 cells/well on monolayers of
MS40-L cells grown in 96-well plates, in the presence of IL-21 (100
ng/mL), IL-10 (100 ng/mL), IL-2 (10 ng/mL), IL-4 (2 ng/mL), and CpG
(1 .mu.M), and cell supernatants screened by ELISA for binding to
H37Rv ManLAM. ManLAM was coated at a concentration of 2 .mu.g/mL in
50 .mu.L of bicarbonate coating buffer per well of a 96 wells ELISA
plate and incubated at 4.degree. C. overnight. The plate was washed
with PBST (0.1% Tween 20 in 1.times.PBS) 4 times, and blocked with
100 .mu.L of 1% BSA in 1.times.PBS for 1 hour at 37.degree. C. 50
.mu.L of the culture supernatant or diluted antibody were added to
the corresponding wells of the ELISA plate containing LAM, and
incubated for 1 hour at 37.degree. C. After additional washing
steps, AP-conjugated goat anti-human IgG (H+L)-antibody was added
to detect bound human antibody. After half an hour of incubation at
37.degree. C., 50 .mu.l of AP-substrate in DEA buffer was added to
the ELISA wells and reactivity was determined by measuring yellow
color at 405 nm. Only 1 out of 78 wells gave a positive signal when
probed with a secondary goat anti-human IgG, IgA, IgM, kappa chain
reagent. After expansion this well was transduced for BCL6 and
Bcl-xL, linked by the self-cleaving porcine teschovirus-1 (P2A)
peptide sequence and followed by a GFP reporter gene is driven by
IRES. These two genes stabilize memory B cells for long-term
replication, and allow the cells to be cultured even after
selection of antigen positive cells by engagement of the BCR. The
retroviral vectors were pseudotyped with Gibbon ape leukemia virus
(GaLV) envelope glycoprotein with the R peptide deleted from the
C-Terminal.TM. domain. Successful transduction of primary B cells
led to expression of BCL-6, Bcl-xL and the marker protein GFP.
Viral titers were determined by counting GFP positive 293T cells
under the fluorescent microscope. Activated B cells were transduced
with retroviral vector in the presence of
polybrene/retronectin.
After further expansion, the transduced cells were subcultured at
limiting dilutions in the presence of IL-21 (100 ng/mL) and IL-2
(long/mL). Well B9 on plate 30 (P30B9) was selected based on its
strong LAM-binding activity and microscopic demonstration of the
presence of a single clone. The P30B9 supernatant bound exclusively
to wells coated with H37Rv-LAM, and not with wells coated with LAM
derived from mycobacterium smegmatic or alpha crystallin. The cells
from this well were lysed and RNA isolated using the RNeasy mini
kit (Qiagen) followed by cDNA synthesis with oligo (dT), using the
superscript III cDNA synthesis system (Invitrogen). Antibody heavy
and light chain variable regions were amplified by using
Smith-Tiller's primers, and cloned in human heavy and light chain
expression vectors. The heavy chain variable region was initially
cloned into a standard IgG vector. However, when combined with the
light chain sequence cloned into a human kappa chain expression
vector no LAM-binding activity was detected. At that point, the
ManLAM-reactive antibodies produced in the original stably
transduced polyclonal well were re-probed with isotype-specific
reagents, and found to be exclusively IgM. The P30B9 VH sequence
was subsequently cloned into an IgM H chain constant region
expression vector, and good binding activity was obtained upon
co-transfection with the corresponding kappa chain.
3. Characterization of Epitope Specificity of A194-01 IgG and P30B9
IgM and Murine Anti-LAM Antibodies Against LAM
A. To define the epitopes recognized by A194-01 IgG and P30B9 IgM,
the binding activities of said antibodies were compared to those of
a number of murine LAM-specific monoclonal antibodies (CS-35,
CS-40, FIND25, FIND170, and the 900 series of monoclonal antibodies
represented by 908.1) against a series of 25 glyconjugates in which
synthetic glycans representing different structures present in LAM
were conjugated to bovine serum albumin (FIG. 4A). These ranged in
size from 4 to 26 carbohydrate rings and represented the range of
structural motifs known to be present in various mycobacterial
LAMs, including a number of poly-arabinose structures both uncapped
and capped with phosphoinositol, alpha(1.fwdarw.2)-linked mono, di-
and tri-Manp mannose structures, and
5-deoxy-5-methylthiopentofuranosyl (MTX) motifs and various capped
Ara4 and Ara6 structures.
Six distinct reactivity patterns were obtained with this antigenic
panel for these monoclonal antibodies (FIG. 4B). The relative
affinities of the monoclonal antibodies for these antigens were
indicated by the titration profile; high affinity reactions retain
high reactivity at the intermediate dilution, whereas low affinity
is indicated by a rapid drop in reactivity. The broadest pattern
was seen for mouse mAb CS-35, which reacted with modest affinity
with LAM derived from Mycobacterium tuberculosis and LAM derived
from Mycobacterium smegmatis, and recognized both capped or
uncapped structures containing the basic Ara4 and Ara6 motifs,
consistent with the known specificity of this mAb for the
.beta.-D-Araf-(1.fwdarw.2)-.alpha.-D-Araf-(1.fwdarw.5)-.alpha.-D-Araf-(1.-
fwdarw.5)-.alpha.-D-Araf motif.
The human monoclonal anti-LAM antibody A194-01 IgG also recognized
a large fraction of these structures and in many cases possessed
the strongest affinity. A109-01 IgG bound strongly to all uncapped
Ara4 and Ara6 structures and to the phosphoinositol-capped Ara4
structure, and less strongly to a subset of the mannose-capped
structures. A109-01 IgG bound well with mono-mannose capped
structures, but very weakly with the di- and tri-mannose
structures, although reactivity with the latter structures was
enhanced when the MTX substitution was present. Four of the 900
series of mouse monoclonal antibodies (represented by 908.1)
reacted with relatively weak affinity with all uncapped Ara4 and
Ara6 structures, but not with any of the capped structures. Two
mouse monoclonal antibodies from FIND (FIND25, also referred to as
KI25), bound strongly with all Ara6 structures, irrespective of the
presence of absence of capping, but did not recognize any Ara4
structures. CS-40, known to react specifically with ManLAM, reacted
weakly with LAM derived from Mycobacterium tuberculosis, and bound
preferentially with mono-mannose-capped Ara4 and Ara6
structures.
The human monoclonal anti-LAM antibody P30B9 IgM, reacted strongly
and with high specificity with ManLAM derived from Mycobacterium
tuberculosis and with dimannose-capped Ara4 and Ara6 structures,
and with considerably weaker activity to the other
mannose-containing structures. Visualization of this residual
activity is dependent on the assay conditions, and shows up in some
assay formats (e.g., FIGS. 4b, 8) but not in others (e.g., FIGS.
16, 18). Without wishing to be bound by theory, the relative
specificity of P30B9 IgM for di-mannose capped structures is
potentially clinically relevant, since terminal mannosyl units are
known to mediate binding of lipoarabinomannan from virulent strains
of the Mycobacterium tuberculosis-complex to human macrophages,
and, furthermore, di-mannose caps are known to be the dominant
modification of LAM derived from Mycobacterium tuberculosis.
Similar results were obtained when the epitope specificity of the
A104-01 IgG and the P30B9 IgM were further mapped in a microarray
assay against a larger panel of carbohydrate antigens. This panel
included several additional polymannose structures which were
recognized by the P30B9 IgM, but not by any of the other antibodies
tested (FIG. 8). This was consistent with the P30B9 IgM preference
for di-mannose capped Ara4 and Ara6 structures, particularly, but
not necessarily, those containing
Man-.alpha.(1.fwdarw.2)-Man-.alpha.(1.fwdarw.5) linked to the
terminal arabinose. The P30B9 IgM also reacted strongly with a
penta-mannose structure (59. AS-3-71) that contained the
Man-.alpha.(1.fwdarw.2)-Man-.alpha.(1.fwdarw.6) but only weakly to
a similar structure containing the
Man-.alpha.(1.fwdarw.3)-Man-.alpha.(1.fwdarw.6) (50. YB-BSA-18).
Despite its preference for the .alpha.(1.fwdarw.2) linkage, the
P30B9 IgM did not react with AS-2-91, a tetra-mannose structure
that contained the Man-.alpha.(1.fwdarw.2) linkage along with an
additional mannose linked .alpha.(1.fwdarw.6) to the second
mannose. Without wishing to be bound by theory, this suggests that
the specificity of the IgM isotype of P30B9 may require that both
sugars of the dimannose motif not contain any additional
substitutions.
B. A more precise titration to map the fine specificities of these
monoclonal antibodies towards the LAM-derived glycans demonstrated
the critical role of the terminal
.beta.-D-Araf-(1.fwdarw.2)-.alpha.-D-Araf-(1.fwdarw.5) disaccharide
in antibody recognition of Ara4 structures. The Ara4 structure
consists of a
.beta.-D-Araf-(1.fwdarw.2)-.alpha.-D-Araf-(1.fwdarw.5)-.alpha.-D-Araf-(1.-
fwdarw.5)-.alpha.-D-Araf tetrasaccharide, while the Ara6 structure
contains an additional
.beta.-D-Araf-(1.fwdarw.2)-.alpha.-D-Araf-(1.fwdarw.3) disaccharide
branch at the second sugar. Three of the monoclonal antibodies
bound to both Ara4 and Ara6 structures independent of mannose
capping. All three monoclonal antibodies bound to the Ara4
structure (YB-8-099) and to YB-BSA-03, corresponding to the Ara4
structure with four additional .alpha.-D-Araf-(1.fwdarw.5) sugars
at the reducing end (FIG. 9A). However, none of the monoclonal
antibodies bound to a related octasaccharide (MJ-LZ-2) that
contained a terminal
.beta.-D-Araf-(1.fwdarw.2)-.alpha.-D-Araf-(1.fwdarw.3)
disaccharide, corresponding to the lower branch of the Ara6
structure. This indicated that the upper branch of the Ara6
structure containing the
.beta.-D-Araf-(1.fwdarw.2)-.alpha.-D-Araf-(1.fwdarw.5) linkage was
recognized by these monoclonal antibodies, and not the lower branch
that contained the
.beta.-D-Araf-(1.fwdarw.2)-.alpha.-D-Araf-(1.fwdarw.3)
disaccharide.
The role of the terminal .beta.-D-Araf-(1.fwdarw.2) linkage in
antibody recognition was examined by probing the reactivity of
these monoclonal antibodies and the Ara6-dependent FIND25 antibody
to three related poly .alpha.-D-Araf-(1.fwdarw.5) structures that
contained truncated forms of the terminal disaccharide (FIG. 9B).
All three structures also contained an internal
.alpha.-D-Araf-(1.fwdarw.3) branch. YB-BSA-07 terminated in a
linear .alpha.-D-Araf-(1.fwdarw.5) structure, and was completely
unreactive with all of the anti-LAM antibodies. YB-BSA-09 contained
additional .alpha.-D-Araf sugars attached via a (1.fwdarw.3) branch
at the penultimate sugars of the two longer branches, resembling
the structure of the Ara6 branch. This structure was recognized
only weakly by the higher concentrations tested of the IgG isotype
of A194-01 and by CS-35. YB-BSA-10 included terminal
.beta.-D-Araf-(1.fwdarw.2) sugars at each of the branches, forming
two complete Ara6 structures at the non-reducing ends of the
polysaccharide. This structure was recognized by all of the
monoclonal antibodies, with relative binding strengths consistent
with their affinities towards the natural LAM antigen. These assays
indicated that a terminal
.beta.-D-Araf-(1.fwdarw.2)-.alpha.-D-Araf-(1.fwdarw.5) disaccharide
was a critical component of all of the available Arabinose-reactive
LAM-specific monoclonal antibodies.
C. A critical distinction between pathogenic strains of the
Mycobacterium tuberculosis-complex such as Mycobacterium
tuberculosis and Mycobacterium bovis and non-pathogenic rapidly
growing strains such as Mycobacterium smegmatis is the presence of
mannose-capped termini on the pathogenic strains. As such,
monoclonal antibodies that are specific for distinct mannosylated
structures could be useful for structural studies and for
determining the functional contributions of these modifications.
The activities of two of the monoclonal antibodies characterized in
this study, CS-35 and FIND25/170, were completely unaffected by the
presence or absence of mannose caps. Binding of the 900 series of
monoclonal antibodies on the other hand was completely abrogated by
mannosylation of any sort (FIG. 4).
CS-40 on the other hand, bound only weakly with the unmodified Ara4
glycan (YB-8-099) but strongly with Ara4 (YB-8-101) and Ara6
(YB-8-149) structures that contained single mannose caps. This
experiment used a modified CS-40 in which the mouse heavy chain
domain was substituted with the human IgG1 constant sequence, since
this resulted in more sensitive detection of binding compared to
the natural mouse antibody used in FIG. 4. The weak reactivity of
CS-40 with the uncapped arabinofuranose structure was reflected in
its weak reactivity with M. smegmatis LAM, compared to M.tb LAM.
Attachment of an .alpha.(1.fwdarw.4) linked MSX sugar to the
terminal mannose (i.e., YB-8-141 and YB-8-149) had no effect on
binding affinity, whereas attachment of a second
.alpha.(1.fwdarw.2) linked mannose sugar (YB-8-111 and YB-8-125) to
generate a dimannose cap completely abrogated CS-40 reactivity
(FIG. 16).
A194-01 possessed a more complex reactivity pattern. A194-01 bound
strongly with uncapped arabinofuranosyl side chains and with
mono-mannose capped Ara4 (YB-8-101) and Ara6 (YB-8-123) structures,
but this mAb reacted only weakly with the dimannose-capped Ara4
(YB-8-111) and even more poorly with tri-mannose capped Ara4
(YB-8-113) and almost not at all for dimannose-capped Ara6
(YB-8-125). As was seen for CS-40, MTX substitution to the
monomannose structures (YB-8-141, YB-8-149) did not inhibit binding
of A104-01, and of particular interest, MSX addition significantly
improved recognition of the dimannose- and trimannose-capped Ara4
structures (YB-8-133, YB-8-143). Consistent with the high
selectivity of P30B9 for ManLAM, the mAb bound specifically with
dimannose-capped Ara4 (YB-111) and Ara6 (YB-8-125) structures. In
contrast to the benign or beneficial effects of MSX substitution on
binding of CS-40 and A194-01, this substitution resulted in the
complete loss of reactivity of P30B9, as did addition of an
additional mannose to form the trimannose capped structures. These
results suggested that the different monoclonal antibodies
recognized different regions and structural aspects of LAM
structure, with some binding solely to the arabinofuranose side
chains and others binding with different levels of specificity to
the capping motifs.
The relative binding specificities and affinities of the
Ara6-reactive antibodies were compared for representative
glycoconjugates (FIG. 11) The overall patterns were consistent with
those obtained for the natural antigens, PILAM and ManLAM (FIG. 3)
and in the preliminary titration against the glycoconjugates (FIG.
4). The human A194-01 IgG possessed higher relative affinity for
all of the uncapped structures and for the MSX-substituted
Ara6-monomannose structure (YB-8-149), reacted with equal affinity
with the Ara6 structure with single mannose caps, but did not
recognize the structures with di-mannose or tri-mannose caps.
FIND25 bound with similar or slightly higher affinity than CS-35 to
all structures that bore the standard Ara6 structure, both in
capped or uncapped forms, but did not bind to two structures
(YB-BSA-06 and YB-BSA-08) in which one of the branches was extended
at the non-reducing end away from the branching point. 908.1 bound
with weaker affinity to all of the uncapped structures, including
the latter two, but did not recognize any of the mannose capped
structures4.
Competition Studies Involving Anti-LAM Monoclonal Antibodies A,
Overview
The ability of individual antibodies to compete for binding of
biotinylated probe mAbs to LAM was titered by ELISA. Typical
competition curves are shown in FIG. 16 for four of the anti-LAM
antibodies, A194-01, CS-35, FIND25 and P30B9. As expected, the
biotinylated antibodies were all competed by their excess amounts
of their unlabeled versions. Murine anti-LAM antibody 908.6
competed poorly, if at all, against the other antibodies. This was
due to some extent to the weak affinity of this antibody, but also
reflects the restriction of 908.6 binding to uncapped structures,
and suggests that capped structures were the dominant targets in
ManLAM recognized by CS-35 and FIND25.
In agreement with its broad reactivity, CS-35 competed fully for
binding of all of the probe antibodies, although its competition
with biotinylated A194-01 was less potent than A194-01 for itself,
consistent with a lower affinity of CS-35 for LAM. Whereas CS-35
competed fully against biotinylated FIND25, FIND25 competed only
partially against labeled CS-35 (.about.74% maximum competition),
and even less effectively against A194-01 (.about.50%). Without
wishing to be bound by theory, this result presumably reflects the
presence of Ara4 structures that are recognized by A194-01 and
CS-35, but not by FIND25, which binds exclusively to the Ara6
motif. The fact that FIND25 competed with the majority of CS-35
binding suggested that Ara6 structures were more common than Ara4
structures. Despite its high affinity, A194-01 competed only
against itself, but not against either CS-35 or FIND25, further
suggesting that the targets in LAM recognized by the latter two
antibodies predominantly consisted of structures (e.g. di-mannose
and tri-mannose-capped structures) that are not recognized by
A194-01. In contrast to this result, A194-01 did compete fully and
efficiently for binding of FIND25 to the un-mannosylated PILAM,
consistent with the role for efficient mannose capping of Ara6
structures in the lack of competition in ManLAM.
Competition studies using the antibody P30B9 further supported the
conclusion that the great majority of Ara6 structures in ManLAM
were capped with di-mannose, and that the bulk of dimannose caps
resided on Ara6 structures. P30B9 competed with .about.70% of
binding of FIND25 and .about.80% of the binding of CS-35 to ManLAM,
confirming that the majority of the structures recognized by these
mouse mAbs were also recognized by P30B9. P30B9 binding to ManLAM
was competed efficiently by itself, and by both CS-35 and FIND25.
The level of competition of P30B9 by FIND25 was close to 100%,
indicating that essentially all of the dimannose-dependent P30B9
binding sites were located on Ara6 sites, and few on Ara4
structures. As expected, A194-01 competed very poorly for binding
of P30B9 to ManLAM, and 908.7 did not compete at all, consistent
with the poor recognition of dimannose-capped structures by these
antibodies. The inability of the latter antibodies to compete
efficiently for binding of P30B9 confirmed that this effect
required binding of the competing mAb to the same branch as the
probe mAb, and that binding to heterologous epitopes located on an
adjacent branch of the same molecule did not lead to effective
competition.
B. Relative A194-01 IgG and P30B9 IgM Affinities by Competition
Assays
Mapping the reactivity of individual monoclonal anti-LAM
antibodies, including the IgG isotype of A194-01 and the IgM
isotype of P30B9, to specific glycan structures allowed for
characterization of the distribution of said specific glycan
structures in LAM by antibody competition studies (FIG. 10). These
competition assays assumed that in order for one antibody to
compete for binding of a second (biotinylated, in cases where they
are from the same species) antibody, the two epitopes must be in
close proximity to each other in the native molecule, potentially,
but not necessarily, on the same or neighboring arabinan branch.
This model was supported by asymmetric competition patterns, where
for example, biotinylated IgG A194-01, which binds to both
uncapped, mono-mannosylated and MSX-substituted Ara4 and Ara6
structures, competed efficiently by itself and by engineered
variants and/or derivatives of A194-01, but only partially by
murine monoclonal antibody FIND25, which binds only to Ara6
structures. On the other hand, murine monoclonal antibody CS-35,
which binds to all Ara4 and Ara6 structures, gives more complete
competition, although less efficiently, presumably due to its
relatively low affinity.
The results of these assays revealed some surprising and unexpected
properties. For example, the IgM isotype of P30B9, which binds to
all di-mannose capped ManLAM structures, was competed strongly and
completely by itself, CS-35 and FIND170. Without wishing to be
bound by theory, the efficient competition of P30B9 by murine
monoclonal anti-LAM antibody FIND25 suggests that the di-mannose
capped structures in native LAM are largely localized to the Ara6
structures recognized by the FIND antibodies, and not appreciably
expressed on Ara4 structures. This heightens the importance of
being able to target and specifically bind to di-mannose capped
Ara6 residues, as di-mannose capping is believed to be the dominant
form of LAM found in virulent strains of the Mycobacterium
tuberculosis-complex. The highly efficient competition of
biotinylated FIND25 by the engineered variant IgM isotype of
A194-01 is further evidence for the increased recognition of
mannosylated structures by the IgM isotype of A194-01.
C. Competition Studies of A194-01 IgG and P30B9 IgM and Murine
Anti-LAM Antibodies to ManLAM and PILAM
Binding competition assays between different anti-LAM monoclonal
antibodies were used to analyze the distribution of various
structural forms in LAM. The IgG isotype of A194-01 recognized both
unmodified Ara4 and Ara6 side chains or chains that contained a
single mannose cap, but did not bind to side chains with either
dimannose or trimannose capping motifs. The two FIND murine
antibodies reacted with all forms of Ara6, but not with any Ara4
structures. P30B9 IgM was relatively specific for Ara4 and Ara6
structures that contained dimannose caps.
Consistent with the broadening in reactivity for the A194-01
constructs with increased valencies, these constructs exhibited an
increased potency in antibody competition activity. When tested for
ability to compete for binding of biotinylated A194-01 IgG against
ManLAM, the decameric A194-01 IgM and tetrameric scFv-IgG variants
competed more efficiently that the A194-01 IgG isotype itself (FIG.
11), thus signifying an increased potential therapeutic and
diagnostic utility, while monomeric Fab and scFv forms competed
less effectively (FIG. 1). The dimeric scFv engineered variant
and/or derivative of A194-01 competed equally as well as the
A194-01 IgG isotype.
When the epitope specificity of the engineered variants and/or
derivatives of A194-01 were compared to that of the A194-01 IgG, it
was observed that they possessed broader reactivity (FIG. 14).
Whereas the IgG isotype did not bind appreciably to the di-mannose
(YB-8-123, YB-8-125) and tri-mannose (YB-BSA-113, YB-BSA-13)
substituted structures, the IgM recognized these structures, and
the scFv-IgG form possessed increased activity against some of
these structures as well. Because di-mannose capping, and
especially di-mannose capped Ara6, is the dominant LAM motif in
virulent Mycobacterium tuberculosis, this suggests a potentially
enhanced utility of these engineered forms of A194-01 in
therapeutic and diagnostic applications.
Unlabeled A194-01 IgG competed against binding of biotinylated
A194-01 IgG to LAM derived from either Mycobacterium tuberculosis
(ManLAM) (FIG. 12A) or Mycobacterium smegmatis (PILAM) (FIG. 12D),
whereas murine monoclonal antibodies FIND170 and P30B9 were not
able to compete for A194-01 binding to either antigen (FIG. 12A,
D). This was consistent with the dominant recognition of Ara4
structures that were recognized by the A194-01 IgG isotype but not
by either FIND170, which is specific for Ara6, or P30B9 IgM, which
is dependent on dimannose capping residues. Similarly, A194-01 did
not compete with binding of either biotinylated FIND25 (FIG. 12B)
or P30B9 IgM (FIG. 12C) to ManLAM, consistent with the different
epitope specificities for these antibodies. In contrast to the
inability of A194-01 IgG fto compete for binding of FIND25 to
ManLAM, A194-01 IgG competed strongly with .about.90% of the
binding of FIND25 to PILAM (FIG. 12E), consistent with the known
absence of mannose-capping in PILAM and with the high affinity of
A194-01 for the uncapped Ara4 and Ara6 structures.
In contrast to the inefficient competition by A194-01 IgG, P30B9
IgM competed with .about.80% binding of FIND25, and FIND170
competed almost completely with binding of P30B9 (FIG. 12B, C).
This strongly suggested that the great majority of the Ara6
structures recognized by the FIND murine antibodies possessed
di-mannose caps, and therefore were also recognized by P30B9 IgM,
and that the majority of the dimannose-capped structures recognized
by P30B9 were present on Ara6 structures. This suggests that
di-mannose capped Ara6 is the dominant immunological motif in LAM
motif from virulent Mycobacterium tuberculosis.
D. Additional Competition Studies
Additional competition studies were undertaken to highlight the
fact that LAM is a complex antigen of undefined heterogeneity. The
definition of the different epitope specificities of LAM-reactive
monoclonal antibodies allowed the use of binding competition assays
to examine the distribution of the various epitopes in native LAMs.
The ability of various antibodies to compete for binding of
biotinylated probe monoclonal antibodies to LAM and synthetic
glycoconjugates was titered by ELISA. Typical competition curves
for three monoclonal anti-LAM antibodies, A194-01 IgG, CS-35, and
FIND25, are shown in FIG. 13A. The biotinylated probe monoclonal
antibodies were competed by their unlabeled versions when present
in large excess. The murine monoclonal antibody 908.6 competed
poorly, if at all, against the other antibodies. This was due to
some extent to the weak affinity of this antibody, but also
reflected the restriction of 908.6 binding to uncapped structures,
and further suggested that mannose-capped structures were the
dominant targets in ManLAM recognized by CS-35 and FIND25.
Consistent with its broad reactivity, CS-35 competed for binding of
biotinylated A194-01 IgG, although less efficiently than did
A194-01 IgG itself, consistent with the higher affinity of A104-01
IgG for LAM. CS-35 also competed fully against biotinylated FIND25,
while FIND25 competed only partially against labeled CS-35
(.about.74% maximum competition) and even less effectively against
A194-01 IgG (.about.50%). This result presumably reflects the
presence of Ara4 structures that are recognized by A194-01 IgG and
CS-35, but not by FIND25, which binds exclusively to structures
containing the Ara6 backbone. Despite its overall high affinity to
LAM, A194-01 IgG competed only against itself, but not against
either CS-35 or FIND25. This suggested the sites in LAM recognized
by the murine monoclonal antibodies were dominated by dimannose and
trimannose-capped structures that were not recognized by A194-01
IgG.
Additional competition studies using P30B9 IgM, which binds
specifically to di-mannose capped ManLAM, further supported the
clinically significant conclusion that the great majority of Ara6
structures in ManLAM were capped with di-mannose, and that the bulk
of di-mannose caps resided on Ara6 structures in Mycobacterium
tuberculosis derived ManLAM. P30B9 IgM competed with .about.80% of
binding of FIND25 to ManLAM (FIG. 13B), consistent with the
majority of the Ara6 structures recognized by FIND25 also being
recognized by P30B9 IgM. P30B9 IgM did not compete for FIND25
binding to PILAM, consistent with the absence of the P30B9
dimannose epitope in PILAM, due to the lack of mannosylation in
PILAM. Furthermore, the A194-01 IgG did not compete for binding of
FIND25 to ManLAM, again consistent with the great majority of the
Ara6 structures bearing di-mannose caps, which are not recognized
by the A194-01 IgG. Confirming the role of mannosylation in this
effect, A194-01 IgG competed very efficiently for binding of FIND25
to PILAM, consistent with the high affinity of A194-01 for PILAM
and the absence of mannose capping in this antigen.
Competition data for the binding of biotinylated P30B9 IgM further
supported this conclusion. Binding of biotinylated P30B9 IgM was
competed most efficiently by itself and with equal efficiency by
CS-35 and FIND25, but only weakly and incompletely by A194-01 IgG
(FIG. 13C). The level of competition by FIND25 was close to 100%,
indicating that essentially all of the di-mannose-dependent P30B9
IgM binding sites were located on
References